Applied Biochemistry and Biotechnology (v.87, #3)

Partial least square analysis of lysozyme near-infrared spectra by Shih-Yao B. Hu; Amy Lillquist; Mark A. Arnold; John M. Wiencek (153-163).
Proteins possess strong absorption features in the combination range (5000-4000 cm−1) of the near infrared (NIR) spectrum. These features can be used for quantitative analysis. Partial least squares (PLS) regression was used to analyze NIR spectra of lysozyme with the leave-one-out, full cross-validation method. A strategy for spectral range optimization with cross-validation PLS calibration was presented. A five-factor PLS model based on the spectral range between 4720 and 4540 cm−1 provided the best calibration model for lysozyme in aqueous solutions. For 47 samples ranging from 0.01 to 10 mg/mL, the root mean square error of prediction was 0.076 mg/mL. This result was compared with values reported in the literature for protein measurements by NIR absorption spectroscopy in human serum and animal cell culture supernatants.
Keywords: Near infrared; Fourier transform infrared; calibration; protein; partial least squares; lysozyme; limit of detection

Amiodarone interactions with membrane lipids and with growth of Bacillus stearothermophilus used as a model by Sónia M. L. J. Rosa; Maria C. Antunes-Madeira; Amália S. Jurado; Vítor V. M. C. Madeira (165-175).
The thermophilic eubacterium Bacillus stearothermophilus was used as a model to study the effects of amiodarone (2-butyl-3-[3′,5′diido-4′α-diethyl-aminoethoxybenzoyl]-benzofuran) in lipid organization and in bacterial growth. Effects on the structural order of lipids were assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and of the propionic acid derivative 3-[p-(6-phenyl)-1,3,5-hexatrienyl] phenylpropionic acid (DPH-PA), probing the outer regions of the bilayer. Amiodarone fluidizes bacterial polar lipid bilayers for temperatures below the phase transition midpoint, and orders the fluid phase of the bacterial polar lipids, as evaluated by DPH and DPH-PA. The ordering and disordering effects, which are concentration dependent, are more extensive when detected by DPH relative to DPH-PA. Growth studies performed in parallel revealed that amiodarone inhibits bacterial growth as a function of concentration. Amiodarone concentrations in the range from 1 to 2.5 µM increased the lag time, decreased the specific growth rate, and decreased the final cell density. Furthermore, 3 µM amiodarone completely inhibited growth. These in vivo effects of amiodarone can be related to its ability to perturb the phospholipid bilayer structure, whose integrity is essential for cell function, viability, and growth.
Keywords: Amiodarone; Bacillus stearothermophilus ; fluorescent probes; membrane organization; bacterial growth

Oscillations of exopolymeric composition and sludge volume index in nitrifying flocs by Felipe Martinez; Ernesto Favela-Torres; Jorge Gomez (177-188).
Protein, carbohydrate, and lipid composition of the exopolymer fraction of a nitrifying sludge in steady-state culture was analyzed after dissociation with 50 mM EDTA and dialysis of the nonfilamentous flocs. Steady-state culture was established when the nitrification rate was constant. The nitrification efficiency at that regime was 93%, also constant. In steady state the concentration of exopolymer protein in the nitrifying sludge floc oscillated from 5 (lowest) to 45 (highest) mg/L with a consistent oscillating pattern having a duration period of 10 d each. Carbohydrate and lipid content in the flocs showed no significant variations (30 and 36 mg/L, respectively). Only 20% of the extracellular polysaccharides had molecular weights higher than 10 kDa, suggesting that the floc aggregation depended on smaller fractions of low-molecular-weight carbohydrates. The oscillations in the concentration of exopolymeric protein coincided with parallel variations in the sludge volumetric index (SVI) value (12.2±2.1 mL/g). Analysis of the polymeric substances of the floc and suspended solids corroborated by statistical analysis indicated that the variations in the SVI of the nitrifying nonfilamentous flocs were mainly related to the changes in the exopolymeric protein content.
Keywords: Nitrification; flocs; extracellular substances; sludge volume index

Kinetic study of addition of volatile organic compounds to a nitrifying sludge by Jorge Gomez; Ramon Mendez; Juan M. Lema (189-202).
The effects of different concentrations of several volatile organic compounds (VOC) such as ethanol, acetate, propionate, and butyrate ranging from 0 to 2000 mg/L as well as a mixture of volatile fatty acids (MVFA) at a 4:1:1 (acetate:propionate:butyrate) ratio on the nitrification rate in batch cultures were studied. The results showed that ethanol and acetate were consumed in a mixotrophic way by the nitrifying sludge. At a concentration of 500 mg/L, the nitrification rate of inhibition was different for each compound in which propionate and butyrate were the most inhibitory. At 2000 mg/L the inhibition was 80% with ethanol and 100% with acetate, propionate, and butyrate. With similar concentrations of MVFA, the inhibition was also similar to that in acetate. The effect of the addition of pulses of MVFA at a ratio of 4:1:1 during 14 h on the performance of the continuous nitrifying process with a hydraulic retention time of 3 d was also studied. No inhibition of the nitrification process was observed with pulses of 750–3000 mg of MVFA/L in the input of the reactor. The results in batch cultures suggest that the different degrees of inhibition of the nitrification process were related to the type of organic matter added. The noninhibitory effect of the organic matter in the continuous cultures on the nitrification efficiency of the nitrifying sludge might be related to the feeding pattern.
Keywords: Nitrification; inhibition rate; ethanol; acetate; propionate; butyrate

High photosynthetic productivity of green microalga Chlorella sorokiniana by Masahiko Morita; Yoshitomo Watanabe; Hiroshi Saiki (203-218).
The batch culture of a newly isolated strain of a green microalga, Chlorella sorokiniana, was carried out using a conical helical tubular photobioreactor. The isolate was capable of good growth at 40°C under an airstream enriched with 10% CO2. The maximum photosynthetic productivity was 34.4g of dry biomass/(m2 of installation area · d) (12-h light/12-h dark cycle) when the cells were illuminated with an average photosynthetic photon flux density (photosynthetically active radiation ([PAR] 400–700 nm) simulating the outdoors in central Japan (0.980 mmol photons/[m2·s]). This corresponded to a photosynthetic efficiency of 8.67% (PAR), which was defined as the percentage of the light energy recovered as biomass (394 kJ/[reactor·d]) to the total light energy received (4545 kJ/[reactor·d]). A similarly high photosynthetic efficiency (8.12% [PAR]) was also attained in the combined presence of 10% CO2, 100 ppm of NO, and 25 ppm of SO2. Moreover, good photosynthetic productivity was also obtained under high temperature and high light intensity conditions (maximum temperature, 46.5°C; 1.737 mmol photons/[m2·s]), when simulating the strong irradiance of the midday summer sun. This strain thus appears well suited for practical application for converting CO2 present in the stack gases emitted by thermal power plants and should be feasible even during the hot summer weather.
Keywords: Microalgae; carbon dioxide; global warming; photosynthesis; bioreactor; effective utilization

Toxicity assessment of tamoxifen by means of a bacterial model by Cristina Luxo; Amália S. Jurado; Vítor M. C. Madeira (219-232).
A strain of Bacillus stearothermophilus was used as a model to study physical perturbations induced in the membrane by the cytostatic tamoxifen (TAM). This study was carried out using two lines of criteria: (1) bacterial growth, and temperature growth range, with determination of growth parameters as a function of TAM concentration; and (2) biophysical studies by differential scanning calorimetry (DSC) and by means of two fluorescent probes to evaluate perturbations promoted by the drug on the structural order of bacterial lipid membranes. The inhibition of growth induced by TAM, the structural bilayer disordering, and the shift in the phase transition temperature to a lower range were also determined in the presence of Ca2+, i.e., a natural membrane stabilizer, to elucidate further perturbing effects of TAM on membranes with putative implications in cell toxicity. Growth inhibition promoted by TAM is potentiated by an increase in growth temperature above the optimal range, but attenuated or relieved by the addition of 2.5 mM Ca2+ to the culture medium. Consistently, fluorescence polarization and DSC studies showed that Ca2+ ions (2.5 mM) effectively compensated for the destabilizing effects promoted by TAM in bacterial lipid membranes.
Keywords: Bacillus stearothermophilus ; calcium; tamoxifen; bacterial growth; membrane physical effects

Quaternized wood as sorbent for reactive dyes by Kun-She Low; Chnoong-Kheng Lee; Bee-Foong Tan (233-245).
Various species of local wood modified with N-(3-chloro-2-hydroxypropyl)-trimethylammonium chloride showed sorption enhancement for hydrolyzed Reactive Blue 2 (HRB) compared to the untreated samples. The enthalpy of sorption of HRB on Simpoh (Dillenia suffruticosa) was found to be endothermic. Maximum sorption capacity calculated from the Langmuir isotherm was 250.0 mg/g. Under continuous flow conditions HRB could be successfully removed. Dye removal was a function of bed depth and flow rate. However, the bed depth service time model of Bohart and Adams was not applicable in the HRB-quaternized wood system. The modified wood was applied to a sample of industrial textile effluent, and it was found to be able to remove the color successfully under batch conditions.
Keywords: Wood; chemical modification; biosorption; reactive dyes

Development of an optimized feeding technology for dairy cows by Jennifer J. Morrison; J. Dayre McNally; Adel Navidzadeh; Marc Beauregard (247-264).
We have previously reported on MB-1, a designer protein with potential application in animal nutrition. Having a high content of selected essential amino acids, MB-1 should provide limiting nutrients for animals and promote growth and production. However, the protein was found to have marginal conformational and proteolytic stability, and, thus, strategies for stabilizing MB-1 were elaborated. We discuss the synthesis of MB-1-Cys dimer, a protein with an intermolecular disulfide bridge. This mutant was exposed to Pronase E protease preparation as well as to proteases extracted from ruminal microbes. It was found that in both cases, MB-1-Cys dimer had a better resistance to proteolytic degradation than MB-1. Denaturation and hydrophobic dye binding studies revealed that this enhanced stability was not owing to conformational stabilization, but rather to changes in surface exposure as a consequence of dimerization. In particular, it was found that binding of ANSA to MB-1-Cys dimer was comparable to that observed for native, compact, natural proteins. We discuss the implications of these results for the design of transgenic protein production systems.
Keywords: Agro-biotechnology; protein design; essential amino acids; proteolytic degradation; rumen protease