Effect of Substrate Concentration on Dark Fermentation Hydrogen Production Using an Anaerobic Fluidized Bed Reactor

The effect of substrate (glucose) concentration on the stability and yield of a continuous fermentative process that produces hydrogen was studied. Four anaerobic fluidized bed reactors (AFBRs) were operated with a hydraulic retention time (HRT) from 1 to 8 h and an influent glucose concentration from 2 to 25 g L⁻¹. The reactors were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C with an influent pH around 5.5 and an effluent pH of about 3.5. The AFBRs with a HRT of 2 h and a feed strength of 2, 4, and 10 g L⁻¹ showed satisfactory H₂ production performance, but the reactor fed with 25 g L⁻¹ of glucose did not. The highest hydrogen yield value was obtained in the reactor with a glucose concentration of 2 g L⁻¹ when it was operated at a HRT of 2 h. The maximum hydrogen production rate value was achieved in the reactor with a HRT of 1 h and a feed strength of 10 g L⁻¹. The AFBRs operated with glucose concentrations of 2 and 4 g L⁻¹ produced greater amounts of acetic and butyric acids, while AFBRs with higher glucose concentrations produced a greater amount of solvents.     

Additional Paper Waste in Pulping Sludge for Biohydrogen Production by Heat-Shocked Sludge

Dark anaerobic fermentation is an interesting alternative method for producing biohydrogen (H₂) as a renewable fuel because of its low cost and various usable organic substrates. Pulping sludge from wastewater treatment containing plentiful cellulosic substrate could be feasibly utilized for H₂ production by dark fermentation. The objective of this study was to investigate the optimal proportion of pulping sludge to paper waste, the optimal initial pH, and the optimal ratio of carbon and nitrogen (C/N) for H₂ production by anaerobic seed sludge pretreated with heat. The pulping sludge was pretreated with NaOH solution at high temperature and further hydrolyzed with crude cellulase. Pretreatment of the pulping sludge with 3% NaOH solution under autoclave at 121 °C for 2 h, hydrolysis with 5 FPU crude cellulase at 50 °C, and pH 4.8 for 24 h provided the highest reducing sugar production yield (229.68 ± 2.09 mg/g_TVS). An initial pH of 6 and a C/N ratio of 40 were optimal conditions for H₂ production. Moreover, the supplement of paper waste in the pulping sludge enhanced the cumulative H₂ production yield. The continuous hydrogen production was further conducted in a glass reactor with nylon pieces as supporting media and the maximum hydrogen production yield was 151.70 ml/g_TVS.     

A Superoxide Dismutase Purified from the Rhizome of <Emphasis Type="Italic">Curcuma aeruginosa</Emphasis> Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K _m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V _max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min⁻¹ mg protein⁻¹. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn²⁺and Fe²⁺ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC₅₀ = 14.36 ± 0.15 μg protein/ml).     

The Correlations Between TCA-Glyoxalate Metabolite and Antibiotic Production of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M4018 Grown in Glycerol,Glucose, and Starch Mediums

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5–20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.     

Sensitive fluorescent probes for determination of hydrogen peroxide and glucose based on enzyme-immobilized magnetite/silica nanoparticles

Sensitive fluorescent probes for the determination of hydrogen peroxide and glucose were developed by immobilizing enzyme horseradish peroxidase (HRP) on Fe₃O₄/SiO₂ magnetic core–shell nanoparticles in the presence of glutaraldehyde. Besides its excellent catalytic activity, the immobilized enzyme could be easily and completely recovered by a magnetic separation, and the recovered HRP-immobilized Fe₃O₄/SiO₂ nanoparticles were able to be used repeatedly as catalysts without deactivation. The HRP-immobilized nanoparticles were able to activate hydrogen peroxide (H₂O₂), which oxidized non-fluorescent 3-(4-hydroxyphenyl)propionic acid to a fluorescent product with an emission maximum at 409 nm. Under optimized conditions, a linear calibration curve was obtained over the H₂O₂ concentrations ranging from 5.0 × 10⁻⁹ to 1.0 × 10⁻⁵ mol L⁻¹, with a detection limit of 2.1 × 10⁻⁹ mol L⁻¹. By simultaneously using glucose oxidase and HRP-immobilized Fe₃O₄/SiO₂ nanoparticles, a sensitive and selective analytical method for the glucose detection was established. The fluorescence intensity of the product responded well linearly to glucose concentration in the range from 5.0 × 10⁻⁸ to 5.0 × 10⁻⁵ mol L⁻¹ with a detection limit of 1.8 × 10⁻⁸ mol L⁻¹. The proposed method was successfully applied for the determination of glucose in human serum sample.     

Evaluation of Biogas Production Potential by Dry Anaerobic Digestion of Switchgrass–Animal Manure Mixtures

Anaerobic digestion is a biological method used to convert organic wastes into a stable product for land application with reduced environmental impacts. The biogas produced can be used as an alternative renewable energy source. Dry anaerobic digestion [>15% total solid (TS)] has an advantage over wet digestion (<10% TS) because it allows for the use of a smaller volume of reactor and because it reduces wastewater production. In addition, it produces a fertilizer that is easier to transport. Performance of anaerobic digestion of animal manure–switchgrass mixture was evaluated under dry (15% TS) and thermophilic conditions (55 °C). Three different mixtures of animal manure (swine, poultry, and dairy) and switchgrass were digested using batch-operated 1-L reactors. The swine manure test units showed 52.9% volatile solids (VS) removal during the 62-day trial, while dairy and poultry manure test units showed 9.3% and 20.2%, respectively. Over the 62 day digestion, the swine manure test units yielded the highest amount of methane 0.337 L CH₄ /g VS, while the dairy and poultry manure test units showed very poor methane yield 0.028 L CH₄/g VS and 0.002 L CH₄/g VS, respectively. Although dairy and poultry manure performed poorly, they may still have high potential as biomass for dry anaerobic digestion if appropriate designs are developed to prevent significant volatile fatty acid (VFA) accumulation and pH drop.     

RuO2/Co3O4 thin films prepared by spray pyrolysis technique for supercapacitors

RuO₂/Co₃O₄ thin films with different RuO₂ content were successfully prepared on fluorine-doped tin oxide coated glass plate substrates by spray pyrolysis method, and their capacitive behavior was investigated. Electrochemical property was performed by cyclic voltammetry, constant current charge/discharge, and electrochemical impedance spectra. The capacitive performance of RuO₂/Co₃O₄ thin films with different RuO₂ content corresponded to a contribution from a main pseudocapacitance and an additional electric double-layer capacitance. The specific capacitance of pure Co₃O₄, 15.5%, 35.6%, and 62.3% RuO₂ composites at the current density of 0.2 A g⁻¹ were 394 ± 8, 453 ± 9, 520 ± 10, and 690 ± 14 F g⁻¹, respectively; 62.3% RuO₂ composite presented the highest specific capacitance value at various current densities, whereas 35.6% RuO₂ composite exhibited not only the largest specific capacitance contribution from RuO₂ (C _sp ^RuO2) at the current density of 0.5, 1.0, 1.5, and 2.0 A g⁻¹ but also the highest specific capacitance retention ratio (46.3 ± 2.8%) at the current density ranging from 0.2 to 2.0 A g⁻¹. Electrochemical impedance spectra showed that the contact resistance dropped gradually with the decrease of RuO₂ content, and the charge-transfer resistance (R _ct) increased gradually with the decrease of RuO₂ content.     

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Petiole Explants of Cassia angustifolia Vahl.—a Potential Medicinal Plant

An effective protocol was developed for in vitro regeneration of the Cassia angustifolia via indirect organogenesis from petiole explants excised from 21-day-old axenic seedlings. Organogenic callus were induced on Murashige and Skoog (MS) medium supplemented with 5.0 μM 2,4-dichlorophenoxy acetic acid and 2.5 μM thidiazuron (TDZ). Adventitious shoot regeneration was achieved on MS medium supplemented with 5.0 μM TDZ as it induced 8.5 ± 0.98 shoots in 85% cultures. The number of shoots and shoot length was significantly enhanced when cultures were subcultured on auxin–cytokinin-containing medium. The highest number of shoots (12.5 ± 1.10) and shoot length (4.3 ± 0.20 cm) was recorded on MS medium supplemented with 5.0 μM TDZ and 1.5 μM indole-3-acetic acid. Regenerated shoots were rooted best on MS medium supplemented with 10.0 μM indole-3-butyric acid followed by their transfer to liquid MS filter paper bridge medium. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 70% survival rate. The plants showed normal morphological characteristics similar to the field grown plants.     

Cloning,Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. K in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg⁻¹ at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K _m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced by Na⁺, NH₄⁺, Mg²⁺, Fe²⁺, Fe³⁺, Co²⁺, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.     

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC₅₀) of the test strip under an optical density scanner were calculated to be 0.1 ± 0.01 ng mL⁻¹ and 0.1 ± 0.01 ng mL⁻¹, 0.56 ± 0.08 ng mL⁻¹, and 0.71 ± 0.06 ng mL⁻¹, respectively, the cut-off levels with the naked eye of 1 ng mL⁻¹ and 1 ng mL⁻¹ for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.      

Biogas Production from Anaerobic Co-digestion of Food Waste with Dairy Manure in a Two-Phase Digestion System

Co-digestion of food waste and dairy manure in a two-phase digestion system was conducted in laboratory scale. Four influents of R0, R1, R2, and R3 were tested, which were made by mixing food waste with dairy manure at different ratios of 0:1, 1:1, 3:1, and 6:1, respectively. For each influent, three runs of experiments were performed with the same overall hydraulic retention time (HRT) of 13 days but different HRT for acidification (1, 2, and 3 days) and methanogenesis (12, 11, and 10 days) in two-phase digesters. The results showed that the gas production rate (GPR) of co-digestion of food waste with dairy manure was enhanced by 0.8–5.5 times as compared to the digestion with dairy manure alone. Appropriate HRT for acidification was mainly determined by the biodegradability of the substrate digested. Three-, 2-, and 1-day HRT for acidification were found to be optimal for the digestion of R0, R1, and R2/R3, respectively, when overall HRT of 13 days was used. The highest GPR of 3.97 L/L·day was achieved for R3(6:1) in Run 1 (1 + 12 days), therefore, the mixing ratio of 6:1 and HRT of 1 day for acidification were considered to be the optimal ones and thus recommended for co-digestion of food waste and dairy manure. There were close correlations between degradation of organic matters and GPR. The highest VS removal rate was achieved at the same HRT for acidification and mixing ratio of food waste and dairy manure as GPR in the co-digestion. The two-phase digestion system showed good stability, which was mainly attributed to the strong buffering capacity with two-phase system and the high alkalinity from dairy manure when co-digested with food waste.     

Angiotensin I-Converting Enzyme Inhibitory Proteins and Peptides from the Rhizomes of Zingiberaceae Plants

Ammonium sulphate cut protein extracts, and their pepsin hydrolysates, from the rhizomes of 15 plants in the Zingiberaceae family were screened for their in vitro angiotensin I-converting enzyme inhibitory (ACEI) activity. The protein extract from Zingiber ottensii had the highest ACEI activity (IC₅₀ of 7.30 × 10⁻⁷ mg protein/mL) and was enriched for by SP Sepharose chromatography with five NaCl step gradients 0, 0.25, 0.50, 0.75 and 1 M NaCl collecting the corresponding five fractions. The highest ACEI activity was found in the F75 fraction, which appeared to contain a single 20.7-kDa protein, suggesting enrichment to or near to homogeneity. The ACEI activity of the F75 fraction was moderately thermostable (−20–60 °C), showed >80% activity across a broad pH range of 4–12 (optimal at pH 4–5) and appeared as a competitive inhibitor of ACE (K _i of 9.1 × 10⁻⁵ mg protein/mL). For the pepsin hydrolysates, that from Zingiber cassumunar revealed the highest ACEI activity (IC₅₀ of 0.38 ± 0.012 mg/mL), was enriched to a single active hexapeptide by RP-HPLC with a strong ACEI activity (IC₅₀ of 0.011 ± 0.012 mg/mL) and acted as a competitive inhibitor of ACE (K _i of 1.25 × 10⁻⁶ mg protein/mL).     

Fully automatic flow method for the determination of scavenging capacity against nitric oxide radicals

In the present work, a fluorimetric automatic method based on multisyringe flow injection analysis (MSFIA) was developed for in vitro evaluation of scavenging capacity against nitric oxide (NO) using 4,5-diaminofluorescein (DAF-2) as an NO-selective fluorogenic probe. The MSFIA manifold was assembled to perform the in-line generation of NO and the competitive reaction of putative scavenger molecules and DAF-2 with NO at conditions close to those found in vivo regarding temperature (37°C), pH (7.4), and concentration of NO (less than 1 μM). This approach allowed the evaluation of scavenging capacity against NO by endogenous antioxidant molecules, pharmaceutical compounds, and human plasma. IC₅₀ values were calculated for rutin (1.30 ± 0.02 μM, positive control), cysteine (321 ± 8 μM), reduced glutathione (1106 ± 93 μM), uric acid (134 ± 12 μM), dipyrone (1.36 ± 0.06 μM), and captopril (363 ± 28 μM). A high degree of automation was attained as the successive dilution of antioxidant standard solutions required for IC₅₀ assessment was performed automatically, in a dilution chamber placed in the flow system. A determination throughput of 16 h^-1 and a good precision were attained (relative standard deviation between 1.6 and 9.0%), fostering the application of the proposed method to routine/screening analysis of scavenging capacity against NO.     

New noncellular fluorescence microplate screening assay for scavenging activity against singlet oxygen

In the present study, a new fluorescence microplate screening assay for evaluating scavenging activity against singlet oxygen (¹O₂) was implemented. The chemical generation of ¹O₂ was promoted using the thermodissociable endoperoxide of disodium 3,3′-(1,4-naphthalene)bispropionate (NDPO₂). The detection of ¹O₂ was achieved using dihydrorhodamine 123 (DHR), a nonfluorescent molecule that is oxidizable to the fluorescent form rhodamine 123 (RH). The combined use of a ¹O₂-selective generator and a highly sensitive probe (DHR) was then successfully applied to perform a screening assay of the ¹O₂ scavenging activities of ascorbic acid, penicillamine, cysteine, N-acetylcysteine (NAC), methionine, reduced glutathione (GSH), dihydrolipoic acid, lipoic acid, and sodium azide. All of these antioxidants exhibited concentration-dependent ¹O₂ scavenging capacities. They could be ranked according to observed activity: ascorbic acid> cysteine> penicillamine> dihydrolipoic acid > GSH> NAC> sodium azide> lipoic acid (IC₅₀ values of 3.0 ± 0.2, 8.0 ± 0.7, 10.9 ± 0.8, 25.2 ± 4.5, 57.4 ± 5.9, 138 ± 13, 1124 ± 128, 2775 ± 359 μM, mean±SEM, respectively) > methionine (35% of scavenging effect at 10 mM). In conclusion, the use of NDPO₂ as a selective generator for ¹O₂ and its fluorescence detection by the highly sensitive probe DHR is shown to be a reliable and resourceful analytical alternative means to implement a microplate screening assay for scavenging activity against ¹O₂. Generation and detection of singlet oxygen     

Enhancement of Human γ-Interferon Production in Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Batch Cultivation

Development of inexpensive and simple culture media and appropriate induction conditions are always favorable for industry. In this research, chemical composition and stoichiometric data for γ-interferon production and recombinant Escherichia coli growth were used in order to achieve a simple medium and favorable induction conditions. To achieve this goal, the effects of medium composition and induction conditions on the production of γ-interferon were investigated in batch culture of E. coli BL21 (DE3) [pET3a-ifnγ]. These conditions were considered as suitable conditions for the production of γ-interferon: 2.5× M9 medium, supplemented with a mixture of amino acids (milligram per liter), including glutamic acid 215, aspartic acid 250, lysine 160, and phenylalanine 90, and induction at late-log phase (OD₆₀₀ = 4.5). Under these conditions, dry cell weight of 6 ± 0.2 g/l and γ-interferon concentration of 2.15 ± 0.1 g/l were obtained. Later, without changing the concentration ratio of amino acids and glucose, the effect of increase in the primary glucose concentration on productivity of γ-interferon was investigated. It was found that 25 g/l glucose will result in maximum attainable biomass and recombinant human γ-interferon. At improved conditions, a dry cell weight of 14 ± 0.2 g/l, concentration and overall productivity of γ-interferon 4.2 ± 0.1 g/l and 420 ± 10 mg/l h, respectively, were obtained.     

Significantly Improved Expression and Biochemical Properties of Recombinant Serratia marcescens Lipase as Robust Biocatalyst for Kinetic Resolution of Chiral Ester

A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 × 10⁵ U/l, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 °C and within the pH range of 5.0−6.8, giving temperature and pH optima at 40 °C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca²⁺, Cu²⁺, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C₁₂). Using racemic trans-3-(4′-methoxy-phenyl)-glycidyl methyl ester [(±)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene–water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 °C under shaking condition (160 rpm), affording (−)-MPGM in nearly 100% ee. The K _m and V _max values of the lipase for (±)-MPGM were 222 mM and 1.24 mmol min⁻¹ mg⁻¹, respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.     

Banana Peel: A Potential Substrate for Laccase Production by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> VkJ2.4.5 in Solid-State Fermentation

In solid-state fermentation, among various solid supports evaluated, banana peel was found to be an ideal support and resulted into higher levels of laccase (6281.4 ± 63.60 U l⁻¹) along with notable levels of manganese peroxidase production (1339.0 ± 131.23 U l⁻¹) by Aspergillus fumigatus VkJ2.4.5. Maximum levels of laccase was achieved under derived conditions consisting of 80% of moisture level, 6 days of incubation period, 6% inoculum level, and an aeration level of 2.5 l min⁻¹. A column-tray bioreactor was designed to scale up and economize the enzyme production in three successive cycles of fermentation using the same fungal biomass. Thermal and pH stability profiles revealed that enzyme was stable up to 50°C and at varying pH range from 5–9 for up to 2 h. The apparent molecular weight of laccase was found to be 34 ± 1 kDa. MALDI-TOF/TOF analysis of the protein showed significant homology with maximum identity of 67% to other laccases reported in database.     

Xylanase Production by <Emphasis Type="Italic">Penicillium canescens</Emphasis> on Soya Oil Cake in Solid-State Fermentation

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na₂HPO₄.2H₂O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895 ± 778 U/g (Erlenmeyer flasks) and 9,300 ± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.     

Determination of cyanide in blood by electrospray ionization tandem mass spectrometry after direct injection of dicyanogold

An electrospray ionization tandem mass spectrometric (ESI-MS-MS) method has been developed for the determination of cyanide (CN^–) in blood. Five microliters of blood was hemolyzed with 50 μL of water, then 5 μL of 1 M tetramethylammonium hydroxide solution was added to raise the pH of the hemolysate and to liberate CN^– from methemoglobin. CN^– was then reacted with NaAuCl₄ to produce dicyanogold, Au(CN)₂^–, that was extracted with 75 μL of methyl isobutyl ketone. Ten microliters of the extract was injected directly into an ESI-MS-MS instrument and quantification of CN^– was performed by selected reaction monitoring of the product ion CN^– at m/z 26, derived from the precursor ion Au(CN)₂^– at m/z 249. CN^– could be measured in the quantification range of 2.60 to 260 μg/L with the limit of detection at 0.56 μg/L in blood. This method was applied to the analysis of clinical samples and the concentrations of CN^– in the blood were as follows: 7.13 ± 2.41 μg/L for six healthy non-smokers, 3.08 ± 1.12 μg/L for six CO gas victims, 730 ± 867 μg for 21 house fire victims, and 3,030 ± 97 μg/L for a victim who ingested NaCN. The increase of CN^– in the blood of a victim who ingested NaN₃ was confirmed using MS-MS for the first time, and the concentrations of CN^– in the blood, gastric content and urine were 78.5 ± 5.5, 11.8 ± 0.5, and 11.4 ± 0.8 μg/L, respectively.     

Production and Characterization of Cellulose by <Emphasis Type="Italic">Acetobacter</Emphasis> sp. V6 Using a Cost-Effective Molasses–Corn Steep Liquor Medium

In order to reduce of the manufacturing cost of bacterial cellulose (BC), BC production by Acetobacter sp. V6 was investigated in shaking culture using molasses and corn steep liquor (CSL) as the sole carbon and nitrogen sources, respectively. The highest BC production was obtained with Ca₃(PO₄)₂-treated molasses. Maximum BC yield (2.21 ± 0.04 g/l) was obtained at 5% (w/v) total sugar in molasses. In improved medium containing molasses and CSL, BC production was observed in the medium after 1 day of incubation and increased rapidly thereafter with maximum yield (3.12 ± 0.03 g/l) at 8 days. This value was approximately twofold higher than the yield in the complex medium. Physical properties of BC from the complex and molasses media were studied using Fourier-transform infrared (FT-IR) spectroscopy and X-ray diffractometer. By FT-IR, all the BC were found to be of cellulose type І, the same as typical native cellulose. The relative crystallinity of BC produced in the complex and molasses media were 83.02 and 67.27%, respectively. These results suggest that molasses and CSL can be useful low-cost substrates for BC production by Acetobacter sp. V6 without supplementation with expensive nitrogen complexes such as yeast extract and polypeptone, leading to the reduction in the production costs.