Methanol removal efficiency and bacterial diversity of an activated carbon biofilter

Motivated by the need to establish an economical and environmentally friendly methanol control technology for the pulp and paper industry, a bench-scale activated carbon biofiltration system was developed. This system was evaluated for its performance in removing methanol from an artificially contaminated air stream and characterized for its bacterial diversity over time, under varied methanol loading rates, and in different spatial regions of the filter. The biofilter system, composed of a novel packing mixture, provided an excellent support for growth and activity of methanol-degrading bacteria, resulting in approximately 100% methanol removal efficiency for loading rates of 1–17 g/m³ packing/h, when operated both with and without inoculum containing enriched methanol-degrading bacteria. Although bacterial diversity and abundance varied over the length of the biofilter, the populations present rapidly formed a stable community that was maintained over the entire 138-day operation of the system and through variable operating conditions, as observed by PCR–DGGE methods that targeted all bacteria as well as specific methanol-oxidizing microorganisms. Phylogenetic analysis of bands excised and sequenced from DGGE gels indicated that the biofilter system supported a diverse community of methanol-degrading bacteria, with high similarity to species in the genera Methylophilus (β-proteobacteria), Hyphomicrobium and Methylocella (both α-proteobacteria).     

Determination of quinolones in plasma samples by capillary electrophoresis using solid-phase extraction

The potential of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC) have been investigated for the separation and quantitative determination of 10 quinolone antibiotics. The influence of different conditions, such as the buffer and pH of the electrolyte, the surfactant and the ion-pairing agents added to the electrolyte and the organic modifier were studied. A buffer consisting of 40 mM sodium tetraborate at pH 8.1 containing 10% (v/v) methanol was found to be a highly efficient electrophoretic system for separating lomefloxacin, enoxacin, norfloxacin, pipemidic acid, ofloxacin, piromidic acid, flumequine, oxolinic acid, cinoxacin and nalidixic acid. A solid-phase extraction method to remove the sample matrix (pig plasma samples) was developed on a C₁₈ cartridge using a mixture of methanol–water (70:30, v/v). The method is specific and reproducible and mean recoveries were in the range 94.0±4.2% and 123.3±4.1% for pig plasma samples over the range used. A linear relationship between concentration and peak area for each compound in pig plasma samples was obtained in the concentration range 5–20 mg l⁻¹ and detection limits were between 1.1 and 2.4 mg l⁻¹.     

Automated surface area measurement of cultured cardiac myocytes

An automated method for rapidly measuring surface area of individual cardiac myocytes was used as an index of myocyte growth. Hearts from 2- to 4-day-old rats were digested by overnight incubation in cold trypsin solution. Enriched suspensions of myocytes were plated at 2×10⁵ cells/well in 12-well-culture plates. Cells were grown in M199 supplemented with 1%, 10% serum or 10% serum plus 10^–7 M norepinephrine. On days 1–4 after plating, cells were fixed in Bouin's Solution and stained with Weigert's Iron Hematoxylin and Biebrich Scarlet-Acid Fuchsin. An inverted microscope, video camera and monitor were coupled to a video image processor (Image Technology Corp.). The enhanced image of stained heart cells was digitized, and perimeter, length, width and area of each selected cell were calculated. One hundred randomly selected cells were measured in each of eight wells from each treatment-day group. Areas of individual myocytes varied widely in culture dishes and the distribition was skewed toward larger cells. The standard deviation increased in proportion to an increase in mean cell area. A logarithmic transformation of the data normalized the data and yielded a more homogeneous variance. The geometric mean area of heart cells supplemented with 1% serum increased only slightly, but significantly, during four days in culture. Geometric mean area of cells supplemented with 10% serum increased nearly four-fold. Supplementing cells with norepinephrine (10^–7 M) in addition to 10% serum did not induce a further increase in cell size. This technique has the potential to rapidly and objectively monitor heart cell growth following pharmacological or toxicological treatments.     

Biofiltration of waste gases containing a mixture of formaldehyde and methanol

Several biofilters and biotrickling filters were used for the treatment of a mixture of formaldehyde and methanol; and their efficiencies were compared. Results obtained with three different inert filter bed materials (lava rock, perlite, activated carbon) suggested that the packing material had only little influence on the performance. The best results were obtained in a biotrickling filter packed with lava rock and fed a nutrient solution that was renewed weekly. A maximum formaldehyde elimination capacity of 180 g m^–3 h^–1 was reached, while the methanol elimination capacity rose occasionally to more than 600 g m^–3 h^–1. Formaldehyde degradation was affected by the inlet methanol concentration. Several combinations of load vs empty bed residence time (EBRTs of 71.9, 46.5, 30.0, 20.7 s) were studied, reaching a formaldehyde elimination capacity of 112 g m^–3 h^–1 with about 80% removal efficiency at the lowest EBRT (20.7 s).     

Control of recombinant human endostatin production in fed-batch cultures of Pichia pastoris using the methanol feeding rate

Endostatin is a 20 kDa carboxyl-terminal fragment of collagen XVIII that strongly inhibits angiogenesis and tumor growth. The methylotrophic yeast, Pichia pastoris, is a robust expression system that can be used to study methods to improve the yields of rhEndostatin. We expressed rhEndostatin in P. pastoris under the control of the alcohol oxidase 1 (aox 1) promoter (Mut⁺ phenotype) as a model, and used a cell biomass of about 50 g l^–1 dry cell wt as a starting point for the induction phase and varied the methanol feed rate at 8 ml l^–1 h^–1, 11 ml l^–1 h^–1 and 15 ml l^–1 h^–1. While the cell growth rate was proportional to the rate of methanol delivery, protein production rate was not. These findings could be used to guide parameters for large-scale production of recombinant proteins in the P. pastoris system.     

Continuous culture of Candida boidinii variant 60 growing on methanol

Summary A new variant, Candida boidinii variant 60, which is less sensitive to methanol and formaldehyde shocks was grown in continuous cultures with methanol as sole carbon source. The substrate concentration in the feeding medium was either 1% methanol or 3% methanol. Biomass production, methanol consumption, the formation of formaldehyde and gas exchange were measured at different dilution rates. With low methanol feeding (10 g/l) maximal productivity of 0.44 g biomass/l·h is obtained at a dilution rate of 0.14 h^–1. Maximal specific growth rate is 0.18 h^–1. A yield of 0.32 g biomass/g methanol was obtained and the respiration quotient was determined as 0.55. Independently of initial substrate concentration, biomass decreases if methanol and formaldehyde are accumulating in the culture broth.In the culture with high methanol feeding (30 g/l) cell concentratioon increases up to 9 g/l at D=0.04 h^–1. At higher dilution rates methanol and form-aldehyde appear in the medium. Formaldehyde is then preferably oxidized without energy advantages for the cells. It seems that this enables the cells to overcome toxic effects caused by methanol and formaldehyde.     

A novel thermotolerant methylotrophicBacillus sp. and its potential for use in single-cell protein production

A thermotolerant methylotrophicBacillus sp. (KISRI TM1A, NCIMB 40040), isolated from the Kuwaiti environment and belonging to the group II spore-forming, bacilli, could not be correlated with any knownBacillus sp. It may, therefore, be a new species. It grew at temperatures from 37° to 58°C from pH 6.5 to 9.0 and on methanol up to 40 g l^–1. It grew well in a chemostat. Its biomass yield coefficient was improved by about 30% by optimization of medium and growth conditions, reaching a maximum of 0.44g g^–1 at 45°C pH 6.8 to 7.0, dilution rate 0.25 h^–1 with methanol at 10 g l^–1. Average crude protein and amino acid content were 84% and 60%, respectively, and maximum productivity attained under laboratory conditions was 5.06 g l^–1h^–1. It was concluded that this strain has good potential for use in single-cell protein production.     

Effects of alternative carbon sources on biological transformation of nitrophenols

The removal of nitrophenols under denitrifying conditions was studied in bench-scale upflow anaerobic sludge blanket (UASB) reactors (R1, R2, R3 and R4) using three different carbon sources. Initially acetate was used as carbon source (substrate) in all the four reactors followed by glucose and methanol. Reactor R1 was kept as control and R2, R3, R4 were fed with 30 mg/l concentration of 2-nitrophenol (2-NP), 4-nitrophenol (4-NP), and 2,4-dinitrophenol (2,4-DNP), respectively. Throughout the study the hydraulic retention time (HRT) and COD/NO₃ ^-–N ratio were kept as 24 h and 10, respectively. 2-Aminophenol (2-AP), 4-aminophenol (4-AP) and 2-amino,4-nitrophenol (2-A,4-NP) were found as the major intermediate metabolites of 2-NP, 4-NP and 2,4-DNP degradation, respectively. Methanol was found to be a better carbon source for 4-NP and 2,4-DNP degradation as compared to acetate and glucose, while 2-NP degradation was not influenced much by the change of substrate. Nitrate nitrogen removal was always more than 99%. COD removal efficiency of the nitrophenol fed reactors varied from 85.7% to 97.7%. The oxidation-reduction potential (ORP) inside the reactors dropped, up to –300 mv, with glucose as carbon source. As the reactors were switched over to methanol, ORP increased to –190 mv. The granular sludge developed inside the reactors was light brown in colour when acetate and glucose were used as substrate, which turned dark brown to black at the end of methanol run. Biomass yield in terms of volatile suspended solids was observed as 0.15, 0.089 and 0.14 g per gram of COD removal for acetate, glucose and methanol, respectively.     

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml⁻¹ for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.     

Continuous anaerobic conversion of sugar beet pulp to biogas

Summary A continuous two step anaerobic digestion of sugar beet pulp as carbon and energy source was carried out. The loading rates in the acidification reactor were varied from 5 g·1^–1·d^–1 to 15 g·1^–1·d^–1 while the hydraulic retention time was in the range of 10 hours to 30 hours; the corresponding values for the methane reactor varied from 2 days to nearly 6 days.With this reactor configuration a biogas yield of more than 85% of the theoretical value for total carbon conversion was achieved resulting in a corresponding COD reduction.     

Benithic-pelagic coupling of nutrient metabolism along an estuarine eutrophication gradient

The shallow, brackish (11–18% salinity) Roskilde Fjord represents a eutrophication gradient with annual averages of chlorophyll, ranging from 3 to 25 mg chl a m^–3. Nutrient loadings in 1985 were 11.3–62.4 g N m^–2 yr^–1 and 0.4–7.3 g P m^–2 yr^–1. A simple one-layer advection-diffusion model was used to calculate mass balances for 7 boxes in the fjord. Net loss rates varied from –32.2 to 17.9 g P m^–2 yr^–1 and from –3.3 to 66.8 g N m^–2, corresponding to 74% of the external P-loading and 88% of the external N-loading to the entire estuary.Gross sedimentation rates measured by sediment traps were between 7 and 52 g p m^–2 yr^–1 and 50 and 426 g N M^–2 yr^–1, respectively. Exchangeable sediment phosphorus varied in annual average between 2.0 and 4.8 g P m^–2 and exchangeable sediment nitrogen varied from 1.9 to 33.1 g N m–1. Amplitudes in the exchangeable pools followed sedimentation peaks with delays corresponding to settling rates of 0.3 m d^–1. Short term nutrient exchange experiments performed in the laboratory with simultaneous measurements of sediment oxygen uptake showed a release pattern following the oxygen uptake, the changes in the exchangeable pools and the sedimentation peaks.The close benthic-pelagic coupling also exists for the denitrification with maxima during spring of 5 to 20 mmol N m^–2 d–1. Denitrification during the nitrogen-limited summer period suggests dependence on nitrification. Comparisons with denitrification from other shallow estuaries indicate a maximum for denitrification in estuaries of about 250 µmol N m^–2 h^–2 achieved at loading rates of about 25–125 g N m^–2 yr^–1.     

Fosfomycin determination in serum by capillary zone electrophoresis with indirect ultraviolet detection

Capillary zone electrophoresis with indirect ultraviolet detection was used for the determination of fosfomycin in serum. Running buffer consisted of a mixture of 200 mM sodium borate with 10 mM phenylphosphonic acid used as ultraviolet absorbing background electrolyte. Relationships between the pH of the buffer and the efficiency of the separation (migration times and selectivities) or the sensitivity of detection were investigated. The method was then validated over a 10–100 μg ml⁻¹ concentration range to be applied to further therapeutic drug monitoring. The choice of ethylphosphonic acid as internal standard is discussed. The specificity and the linearity of the technique are demonstrated. The inter-day precision was satisfactory with a relative standard deviation of less than 2%. Accuracy was calculated with a standard error near 0.5 and 18% for 100 and 10 μg ml⁻¹, respectively.     

Dextrin and salt as impurities of histological dyes

Summary The dextrin and salt contents of 18 common histological dyes (usually obtained from more than one supplier) were estimated by solvent extraction of dye from the commercial sample with organic solvents such as ethanol, iso-propanol and methanol. The procedure is also suitable for preparing large quantities of dextrin and salt-free dyes. The dye contents of the commercial dye samples varied widely:many commercial dyes were pure but nearly half the samples contained less than 75% dye. The solvent insoluble residues from 10 commercial pyronin and rhodamine dyes were studied in more detail. Tests for dextrin and for NH ₄ ⁺ , Cl^– and SO ₄ ^2– ions were applied and the dextrin:salt ratio determined by combustion. Only 3 of the residues contained appreciable amounts of salt. Certain samples contained organic materials other than dextrin.     

Naphthenic acids affect plant water conductance but do not alter shoot Na+ and Cl− concentrations in jack pine (Pinus banksiana) seedlings

Solution culture-grown, six-month old jack pine (Pinus banksiana Lamb.) seedlings were treated with naphthenic acids (NAs) (150 mg l^–1) and sodium chloride (45 mM NaCl) which were applied together or separately to roots for four weeks. NAs aggravated the effects of NaCl in inhibiting stomatal conductance (g _s) and root hydraulic conductance (K_r). Naphthenic acids did not affect needle and root electrolyte leakage in the absence of NaCl. However, in plants treated with NaCl, NAs further increased electrolyte leakage from needles and NaCl induced electrolyte leakage from needles, but not from roots. Both NaCl and NAs treatments resulted in a reduction in root respiration. The measured Na⁺ and Cl^– concentrations in the shoots for combined NaCl + NAs treatments were lower than in NaCl-only treatments. These decreases were correlated with a reduction in water conductance. The accumulation of Na⁺ and Cl^– in shoots was accompanied by an increased in needle electrolyte leakage. However, greater concentrations of Cl^– compared with Na⁺ were present in shoots and in the xylem sap suggesting that roots had relatively lower capacity for Cl^– storage compared with Na⁺.     

Feasibility study on the use of hyperosmolar medium for improved antibody production of hybridoma cells in a long-term,repeated-fed batch culture

To test the feasibility of using hyperosmolar medium for improved antibody production in a long-term, repeated fed-batch culture, the influence of various culture conditions (serum concentration and cultivation method) on the hybridoma cells' response to hyperosmotic stress resulting from sodium chloride addition was first investigated in a batch culture. The degree of cell growth depression resulting from hyperosmotic stress was dependent on serum concentrations and cultivation methods (static and agitated cultures). Depression of cell growth was most significant in agitated cultures with low serum concentration. However, regardless of serum concentrations and cultivation methods used, the hyperosmotic stress significantly increased specific antibody productivity (q _MAb). Increasing osmolality from 284 to 396 mOsm kg^–1 enhanced the q_MAb in agitated cultures with 1% serum by approximately 124% while the similar osmotic stress enhanced the q _MAb in static cultures with 10% serum by approximately 153%. Next, to determine whether this enhanced q_MAb resulting from hyperosmotic stress can be maintained after adaptation, long-term, repeated-fed batch cultures with hyperosmolar media were carried out. The cells appeared to adapt to hyperosmotic stress. When a hyperosmolar medium (10% serum, 403 mOsmkg^–1) was used, the specific growth rate improved gradually for the first four batches and thereafter, remained constant at 0.040±0.003 (average ± standard deviation) hr^–1 which is close to the value obtained from a standard medium (10% serum, 284 mOsmkg^–1) in the batch culture. While the cells were adpating to hyperosmotic stress, the q_MAb was gradually decreased from 0.388×10^–6 to 0.265×10^–6 g cell hr^–1 and thereafter, remained almost constant at 0.272±0.014× 10^–6 g cell^–1 hr^–1. However, this reduced q _MAb after adaptation is still approximately 98% higher than the q_MAb obtained from a standard medium in the batch culture.The authors would like to thank Dr.M. Kaminski for providing the hybridoma cell line used in this study. This work was supported by the Korea Science and Engineering Foundation.     

Heterotrophic glucose uptake and respiration in Lake Kinneret

The turnover times of glucose, averaged for 0–10 m in the upper waters of Lake Kinneret and measured by the addition of single or multiple concentrations of substrate, ranged from 23 to 188 hours and 1 to 87 hours respectively. Potential uptake rates (estimated as V_max) ranged from 0.095 to 1.94 µg glucose l^–1h^–1, while measured uptake rates varied from 0.09 to 1.1 µg glucose l^–1h^–1. Concentrations of dissolved carbohydrates and glucose averaged 0.71 mg glucose equivalents l^–1 and 39 µg glucose l^–1 respectively. No evident relationships between glucose cycling and any fractions of dissolved organic matter, phytoplankton biomass or primary productivity were found. Turnover times were generally most rapid immediately after the decline of the spring Peridinium bloom. The respiration percentage of incorporated glucose ranged from 25% to 61% with highest values during the summer months. Respiration may be influenced by the nature of the indigenous bacterial population as well as by temperature. Daily heterotrophic glucose carbon uptake was about 9% of the photosynthetic incorporation and could provide a bacterial yield of about 7 × 10⁴ ml^–1d^–1.     

Simple and sensitive assay of zonisamide in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A rapid and sensitive method for the assay of zonisamide in serum was developed using a solid-phase extraction technique followed by high-performance liquid chromatography. A 20-μl volume of human serum was first purified with a Bond-Elut cartridge column. Then, the methanol eluate was injected onto a reversed-phase HPLC column with a UV detector. The mobile phase was acetonitrile—methanol—distilled water (17:20:63, v/v) and the detection wavelength was 246 nm. The detection limit was 0.1 μg/ml in serum. The coefficients of variation were 4.2–5.6% and 5.1–9.1% for the within-day and between-day assays, respectively. This method can be used for clinical pharmacokinetic studies of zonisamide in serum even in infant patients with epilepsy.     

Determination of gestrinone in human serum by liquid chromatography–electrospray tandem mass spectrometry

A rapid, sensitive and specific high-performance liquid chromatography–electrospray tandem mass spectrometric method has been developed for the determination of gestrinone (R 2323) in human serum using mifepristone (RU 486) as an internal standard. R 2323 was extracted from human serum by an ether extraction procedure. Multiple reaction monitoring was used to detect R 2323 and RU 486. The calibration curve was linear over the range of 3.5–177 ng/ml (r²≥0.99) with the limitation of detection of 0.8 ng/ml. The intra-day precision and accuracy, expressed as C.V. and RE, ranged from 2.3–13.7 to −4.8–3.0%. The inter-day precision and accuracy ranged from 5.5–14.8 to −6.7–3.1%. The mean recovery was 91.0% for R 2323, and 90.6% for the internal standard. The method was successfully applied to the pharmacokinetic study of R 2323.     

Role of plankton in the turnover of organic matter on the Great Barrier Reef,Australia

Number, biomass and production of phytoplankton, bacteria, micro- and mesozooplankton and turnover of labile and stable organic matter were measured in waters over some Capricornia round reefs, and over the reefs of Lizard Island. Primary production was 10 to 40 mg C m^–3 d^–1 but was lower over the living reefs. Microbial wet biomass in reef waters varied from 100 to 500 mg m^–3, and production from 4 to 68 mg C m^–3 d^–1, which was commensurable with primary production. The biomass of microzooplankton (ciliates, zooflagellates and larvae) in waters of Lizard Island reefs reached 100–300 mg m^–3. Mesozooplankton biomass at night in reef waters of Heron Island varied from 200 to 800 mg m^–3. Its composition depended upon the tide phase. PB coefficients in bacterioplankton were 0.3 to 1.2 per day. The food demand of bacterioplankton in waters over the reefs was 5 to 20 times higher than the primary phytoplankton production. Labile organic matter (LOM) doubled in waters after it stayed over living reef for several hours. The turnover time of LOM in reef waters was as short as 1–2 weeks.     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.