Maximizing the productivity of catalytic biofilms on solid supports in membrane aerated reactors

A new solid support membrane aerated biofilm reactor was designed for the synthesis of enantiopure (S)‐styrene oxide utilizing Pseudomonas sp. strain VLB120ΔC growing in a biofilm as biocatalyst. In analogy to traditional packed bed systems, maximizing the volumetric oxygen mass transfer capability (k_La) was identified as the most critical issue enabling a consistent productivity, as this parameter was shown to directly influence biofilm growth and biotransformation performance. A microporous ceramic unit was identified as an ideal microenvironment for biofilm growth and for efficient oxygen transfer. A uniform and dense biofilm developed on this matrix. Due to this dual function, the reactor configuration could be significantly simplified by eliminating additional packing materials, as used in traditional packed bed reactors. Up to now, a maximum productivity of 28 g L day^?1 was achieved by integrating an in situ substrate feed and an in situ product recovery technique based on a silicone membrane. The system was stable for more than 30 days before it was actively terminated. Biotechnol. Bioeng. 2010;106: 516–527. © 2010 Wiley Periodicals, Inc.     

Bioluminescence of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> HK44 in the course of encapsulation into silica gel. Effect of methanol

The bioluminescence (BLM) and colony-forming units (CFU) of Pseudomonas fluorescens HK44 were monitored during encapsulation into pre-polymerized Si(OMe)₄. The non-induced BLM of free cells was increased in the presence of 0.5–2.5 % MeOH. After mixing silica sol with the cell suspension, both BLM and CFU dropped to 1–3 and 8–18 %, respectively; both remained lowered as long as the silica biofilm contained residual MeOH. The kinetics of MeOH being released from silica biofilms (a thickness of 2–6 mm) were first-order. The decrease of bacterial activity due to encapsulation was proportional to the biofilm thickness. MeOH evolving during encapsulation is probably the principal stress factor but not the only one.     

Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis

Biofilms are known to be robust biocatalysts. Conventionally, they have been mainly applied for wastewater treatment, however recent reports about their employment for chemical synthesis are increasingly attracting attention. Engineered Pseudomonas sp. strain VLB120ΔC biofilm growing in a tubular membrane reactor was utilized for the continuous production of (S)‐styrene oxide. A biofilm specific morphotype appeared in the effluent during cultivation, accounting for 60–80% of the total biofilm irrespective of inoculation conditions but with similar specific activities as the original morphotype. Mass transfer of the substrate styrene and the product styrene oxide was found to be dependent on the flow rate but was not limiting the epoxidation rate. Oxygen was identified as one of the main parameters influencing the biotransformation rate. Productivity was linearly dependent on the specific membrane area and on the tube wall thickness. On average volumetric productivities of 24 g L day^?1 with a maximum of 70 g L day^?1 and biomass concentrations of 45 g_BDW L have been achieved over long continuous process periods (≥50 days) without reactor downtimes. Biotechnol. Bioeng. 2010. 105: 705–717. © 2009 Wiley Periodicals, Inc.     

In situ growth of gold nanoparticles by enzymatic glucose oxidation within alginate gel matrix

In situ growth of gold nanoparticles (Au NPs) was performed in an alginate gel matrix co‐encapsulating Au NP seeds and glucose oxidase (GOx) for the development of a glucose‐sensing platform. We observed a simultaneous growth of Au NPs in the alginate matrix through the reduction of AuCl by H₂O₂ on Au NP seeds. The detection of the glucose level was carried out successfully via the coupling of Au NP enlargement with the oxidation of glucose catalyzed by the immobilized GOx. The enlargement of Au NPs in the alginate matrix exhibited only a red‐shift in absorbance maxima, while the generation of small Au NPs in a free solution caused a blue‐shift in higher glucose concentrations. This study shows that the co‐encapsulation of metal NPs and bioreceptor in a gel matrix may provide a simple route for the fabrication of an optical biosensor. Biotechnol. Bioeng. 2010;105: 210–214. © 2009 Wiley Periodicals, Inc.     

Application of the nearest-neighbor Ising model to the helix-coil transition of polypeptides in mixed organic solvents.

Using the formalism of nearest-neighbor Ising model and assuming that the allowed states for a monomeric unity of a polypeptide chain in solutions containing strong acids are E (helix), C (coil), and CS (solvent-bonded coil), the partition function of the system was deduced analytically. Equations were obtained which permitted the prediction of the characteristic thermodynamic behavior of the helix–coil transition under these conditions. These equations were used to examine critically the possible correlations between experimental data obtained using different techniques. Particular attention was devoted to quantities called “transition enthalpies,” obtained from the slope of the transition curves at the point where the helix fraction is one-half (ΔH), or for measurements of the heat of solution of the polymer over the total range of solvent composition (ΔH), or from heat capacity measurements taken at various temperatures (ΔH). Literature data of ΔH(j = opt, sol, cal) for the system poly-γ-benzyl-L -glutamate in mixtures of dichloroacetic acid and 1,2-dichloroethane were carefully analyzed.     

Construction of Equi-replicated Balanced Block Designs with Block Sizes Exceeding the Number of Treatments

In this note it is shown that the block design with incidence matrix Ñ = [NN… N], where N = c_1hN_h + c_oh (11′–N_h). c_oh and c_1h are any non-negative integers and N_h,h = 1, 2,…,p, are incidence matrices of balanced incomplete block designs with the same number of treatments t, is a balanced block design with the block sizes exceeding the number of treatments. In derivation the matrix M₀, introduced by CALIński (1971) is utilized.     

Equi-replicated balanced block designs generated by a balanced matrix

In this paper it is shown that if N= \documentclass{article}\pagestyle{empty}\begin{document}$ \mathop \sum \limits_{i = 1}^{S_h} $\end{document} c_ihN_ih, where c_ih are some non-negative integer numbers and N_ih are such incidence matrices that A_h = \documentclass{article}\pagestyle{empty}\begin{document}$ \mathop \sum \limits_{i = 1}^{S_h} $\end{document} i N_ih is a balanced matrix defined by SHAH (1959), for h = 1, 2,…, p, then a block design with an incidence matrix Ñ = [N, N,…,N] is an equi-replicated balanced block design. Here the balance of a block design is defined in terms of the matrix M₀ introduced by CALI?SKI (1971).     

Interplay of reactive oxygen species,intracellular Ca2+ and mitochondrial homeostasis in the apoptosis of prostate cancer cells by deoxypodophyllotoxin

The limited treatment option for recurrent prostate cancer and the eventual resistance to conventional chemotherapy drugs has fueled continued interest in finding new anti‐neoplastic agents of natural product origin. We previously reported anti‐proliferative activity of deoxypodophyllotoxin (DPT) on human prostate cancer cells. Using the PC‐3 cell model of human prostate cancer, the present study reveals that DPT induced apoptosis via a caspase‐3‐dependent pathway that is activated due to dysregulated mitochondrial function. DPT‐treated cells showed accumulation of the reactive oxygen species (ROS), intracellular Ca surge, increased mitochondrial membrane potential (MMP, ΔΨ_m), Bax protein translocation to mitochondria and cytochrome c release to the cytoplasm. This resulted in caspase‐3 activation, which in turn induced apoptosis. The antioxidant N‐acetylcysteine (NAC) reduced ROS accumulation, MMP and Ca surge, on the other hand the Ca²⁺ chelator BAPTA inhibited the Ca overload and MMP without affecting the increase of ROS, indicating that the generation of ROS occurred prior to Ca²⁺ flux. This suggested that both ROS and Ca signaling play roles in the increased MMP via Ca‐dependent and/or ‐independent mechanisms, since ΔΨ_m elevation was reversed by NAC and BAPTA. This study provides the first evidence for the involvement of both ROS‐ and Ca‐activated signals in the disruption of mitochondrial homeostasis and the precedence of ROS production over the failure of Ca²⁺ flux homeostasis. J. Cell. Biochem. 114: 1124–1134, 2013. © 2012 Wiley Periodicals, Inc.     

Optimum Designs for Estimating the Variance Components in a Simple Variance Component Model (Model II), I

The methods of optimal designing of experiments proposed by WALD (1943) are used for determination of an A_σ2-optimal concrete design for estimation of σ^2′ = (σ, σ) in case of one-way analysis of variance. Starting point of definition of the optimality criterion is a quadratic loss matrix.     

Einfluß extracellulärer Kaliuminoenkonzentrationen auf die celluläre Natriumionenkonzentration von Lodderomyces elongisporus D

It was found that the cellular Na⁺-concentration (C) of Lodderomyces elongisporus D is depended on the extracellular K⁺-concentration (C). The relationship can be described by an equation in the form The function of the natrium ion seem to be to support the utilisation rate of potassium ion at lower extracellular K⁺-concentration.     

A novel nonwoven hybrid bioreactor (NWHBR) for enhancing simultaneous nitrification and denitrification

This study proposed a nonwoven hybrid bioreactor (NWHBR) in which the nonwoven fabric played dual roles as a biofilm carrier and membrane‐like separation of the flocculent sludge in the reactor. The results of long‐term monitoring demonstrated that the NWHBR could achieve simultaneous nitrification and denitrification (SND), with nearly complete ammonium removal and 80% removal of total nitrogen. The biofilm attached to the nonwoven fabric removed 27% of the chemical oxygen demand (COD) and 36% of the nitrate in the reactor, an enhanced elimination of nutrients that was attributed to the increased mass transfer within the biofilm due to permeate drag. The results of batch experiments showed that the flocculent sludge played a more dominant role in nitrification and denitrification (79% and 61%, respectively) than the biofilm (21% and 36%, respectively). The batch experiments also revealed that the enforced mass transfer, with an effluent recirculation rate of 4.3 L/m²h (which was the same as the flux during the reactor's long‐term operation), improved the denitrification rate by 58% (i.e., from 9.0 to 14.2 mg‐NO‐N/h). Pyrosequencing of the 16S rRNA gene amplification revealed a high microbial diversity in both the flocculent sludge and biofilm, with Proteobacteria, Bacteroidetes and Chloroflexi as the dominant groups. A phylogenetic (P) test indicated that the NWHBR contained phylogenetically distinct microbial communities: those in the biofilm differed from those in the flocculent sludge. However, the communities on the exterior and interior of the biofilm were more similar to each other. Due to its good SND performance, low physical back‐washing frequency and low air‐to‐water ratio, the NWHBR represents an attractive alternative for the wider application of either low‐cost membrane bioreactors or biofilm reactors. Biotechnol. Bioeng. 2013; 110: 1903–1912. © 2013 Wiley Periodicals, Inc.     

A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms

Aims: Research on biofilms requires validated quantitative models that focus both on matrix and viable bacterial mass. In this study, a new microplate model for the detection of Staphylococcus aureus biofilms was developed. Methods and Results: Dimethyl methylene blue (DMMB) dye was used to quantify biofilm matrix colorimetrically. Initially developed for the detection of glycosaminoglycans, the DMMB protocol was optimized for S. aureus biofilm research. In addition, the redox indicator resazurin was used to determine the viable bacterial biofilm burden. Conclusion: A new, simple and reproducible microplate test system based on DMMB and resazurin, offering a reliable differentiation between biofilm matrix and cellular activity, was developed and validated for the detection of S. aureus biofilms. Significance and Impact of the Study: The DMMB–resazurin microtitre plate model is a valuable tool for high capacity screening of biocides and for the development of synergistic mixtures of biocides, destroying both biofilm matrix and bacteria.     

Engineered catalytic biofilms for continuous large scale production of n‐octanol and (S)‐styrene oxide

This study evaluates the technical feasibility of biofilm‐based biotransformations at an industrial scale by theoretically designing a process employing membrane fiber modules as being used in the chemical industry and compares the respective process parameters to classical stirred‐tank studies. To our knowledge, catalytic biofilm processes for fine chemicals production have so far not been reported on a technical scale. As model reactions, we applied the previously studied asymmetric styrene epoxidation employing Pseudomonas sp. strain VLB120ΔC biofilms and the here‐described selective alkane hydroxylation. Using the non‐heme iron containing alkane hydroxylase system (AlkBGT) from P. putida Gpo1 in the recombinant P. putida PpS81 pBT10 biofilm, we were able to continuously produce 1‐octanol from octane with a maximal productivity of 1.3 g L day^?1 in a single tube micro reactor. For a possible industrial application, a cylindrical membrane fiber module packed with 84,000 polypropylene fibers is proposed. Based on the here presented calculations, 59 membrane fiber modules (of 0.9 m diameter and 2 m length) would be feasible to realize a production process of 1,000 tons/year for styrene oxide. Moreover, the product yield on carbon can at least be doubled and over 400‐fold less biomass waste would be generated compared to classical stirred‐tank reactor processes. For the octanol process, instead, further intensification in biological activity and/or surface membrane enlargement is required to reach production scale. By taking into consideration challenges such as biomass growth control and maintaining a constant biological activity, this study shows that a biofilm process at an industrial scale for the production of fine chemicals is a sustainable alternative in terms of product yield and biomass waste production. Biotechnol. Bioeng. 2013; 110: 424–436. © 2012 Wiley Periodicals, Inc.     

Hydrobiological Studies on Suraj Kund and Chandrama Kund,Hot Springs of Rajgir,Bihar, India

The hot water of Rajgir springs is used for drinking and bathing purposes by tourists. Certain physico-chemical characteristics (temperature, pH, NO, PO, etc.) of the water along with phycological parameters viz. community composition, species diversity, standing crop etc. were measured from June 1986 to April 1987. The water was deficient in Na, NO and PO ions. The hot springs were mainly dominated by algae belonging to Cyanophyceae and Bacillariophyceae. The algal community comprised 18 species, with dominance of Cyanophyceae over Bacillariophyceae. While Mastigocladus laminosus and species of Phormidium were dominant in Suraj Kund, species of Oscillaroria and Synechococcus dominated in Chandrama Kund. Diatoms comprised about 10 % of the algal community. Though there was a considerable seasonal change in species diversity of the algal community the total biomass (chlorophyll a extracted per unit area from the algal mat) remained constant.     

Kinetics of ethidium's intercalation in packaged bacteriophage T7 DNA: effects of DNA packing density

The kinetics of ethidium's intercalative binding to DNA packaged in bacteriophage T7 and two T7 deletion mutants have been determined, using enhancement of fluorescence to quantitate binding. At a constant ethidium concentration, the results can be described as first-order binding with two different rate constants, k (= k₁ + k_?1) and k (= k₂ + k_?2). The larger rate constant (k) was at least four orders of magnitude smaller than the comparable first-order forward rate constant for binding to DNA released from its capsid. At 25°C values of k decreased as the amount of DNA packaged per internal volume increased. This latter observation indicates that the rate of ethidium's binding to packaged T7 DNA is limited by an event that occurs inside of the DNA-containing region of T7, not by the crossing of T7 capsid's outer shell. Arrhenius plots of kM are biphasic, indicating a transition for packaged DNA at a temperature of 20°C. The data indicate that k s are limited by either sieving of ethidium during its passage through the packaged DNA or subsequent hindered intercalation.     

Shear‐induced detachment of biofilms from hollow fiber silicone membranes

A suite of techniques was utilized to evaluate the correlation between biofilm physiology, fluid‐induced shear stress, and detachment in hollow fiber membrane aerated bioreactors. Two monoculture species biofilms were grown on silicone fibers in a hollow fiber membrane aerated bioreactors (HfMBR) to assess detachment under laminar fluid flow conditions. Both physiology (biofilm thickness and roughness) and nutrient mass transport data indicated the presence of a steady state mature biofilm after 3 weeks of development. Surface shear stress proved to be an important parameter for predicting passive detachment for the two biofilms. The average shear stress at the surface of Nitrosomonas europaea biofilms (54.5 ± 3.2 mPa) was approximately 20% higher than for Pseudomonas aeruginosa biofilms (45.8 ± 7.7 mPa), resulting in higher biomass detachment. No significant difference in shear stress was measured between immature and mature biofilms of the same species. There was a significant difference in detached biomass for immature vs. mature biofilms in both species. However, there was no difference in detachment rate between the two species. Biotechnol. Bioeng. 2013; 110: 525–534. © 2012 Wiley Periodicals, Inc.     

How Large is the Probability for the Estimate of a Variance Component to be Negative?

For a balanced one-way classification, where the normally distributed observations obey a random model y_ij=μ+b_i+c_ij with two variance components var (b_i) = δ and var (c_ij) = δ, the probability is given that the analysis of variance estimate of δ will be negative. This probability depends on δ/δ and the degrees of freedom in the ANOVA table. Tables for this probability are given. If the normally distributed observations obey an intra-class correlation model, the probability that the Mean Square between groups is smaller than the Mean Square within groups can also be evaluated from the given tables.     

Conformational analysis of acetyl-L-phenylalanine p-acetyl and p-valeryl anilides

Empirical force-field calculations and ir and ¹H-nmr spectra indicate that five-membered (C₅) and seven-membered (C) hydrogen-bonded rings are the preferred conformations of acetyl-L -Phe p-acetyl and p-valeryl anilides in nonpolar media. The C₅/C ratio was found to be dependent on the dryness of the solute and the solvent. This fact and the results from conformational-energy calculations suggest that a molecule of water participates in the stabilization of the C conformation.     

Nmr studies on complex formation of poly(L-lysine HBr) with carbonate ion in aqueous solution

Diemer formation of poly(L -lysine HBr) in carbonate buffer at pD 10.5 was reported in our previous paper [Biopolymers(1981) 20 , 345–357]. The mechanism of the dimer formation was investigated employing carbon-13 and proton nmr. pD dependence of the ¹³C-nmr spectrum of poly(L -lysine HBr) in the presence of carbonate ion clearly shows that a complex formation between the CO ion and protonated ε-amino group is involved in the stabilization of the dimer form. The lifetime of the complex is longer than 10^?2 s. A stoichiometric evaluation suggests that CO bridges two lysyl side chains. A molecular model of the dimer form designated as a single antiparallel β-ribbon was proposed and discussed in the light of hydrodynamic and ir spectroscopic properties reported earlier. Concentration change experiments indicate that CO binds not only to the dimer formation is inferred as stabilization of the single antiparallel β-ribbon as an intermediate structure in the conversion between the α-helix and β-sheet. The α-CH proton signal of the lysine residue located in the ordered structure (α-helix and β-form) was observed to be separate from that in the random-coil region.     

Heat capacity changes and partial molal heat capacities of some di- and tripeptides in water

Integral enthalpies of solution of several dipeptides and tripeptides in water at low concentrations have been determined at 25 and 35°C. These data have been used to derive the changes in heat capacity on dissolution at infinite dilution ΔC at 30°C. Limiting partial molal heat capacities ΔC have been determined by combining ΔC with C_p2 (heat capacity of pure solid peptides). Using the data on ω-amino acids and these peptides, the partial molal heat capacity of a peptide group ? CONH? was semiquantitatively estimated.