Studies on immobilization of Bacteria

Cell immnhilizatinn technology in a rapidly expanding arna in the endeavour of microbial fnrmentatiwn.During the lnmt 15 years anveral prnceafinn have been developed and more are in developmental atage of approaching commercial utilizatinn.In the present programme it was planned to develop an optimized process for the innobilization of alpha amylase producing Bacillus polymyxa (CBTB 25) an isolate obtained from Cochin University campus primarily for the production of alpha-amylase.Optimal concentration of support material that attributaa stability and maximal activity to the immobilized cell beads was determined using different concentrations of sodium aliginate as support and estimation of amylase production.An overeall assessment of the data obtained for the various studies conducted denotes that immobilized cells synthesize alpha-amylase at comparable rates with free cells and produce reducing sugara at a higher level than free cells.Results indicated that both phosphate and citrate buffers could be used for disrupting the immobilized beads since they enforced maximal release of cells through leaching from the beads within one hour.On comparative analysis it was observed that immobilized cells could synthesize alpha amylase at similar levels with free cells of B.polymyxa.On Co-immobilization of B.Polymyxa with S.cerevisiae,the co-immobilizate beads could effeciently convert starch directly to ethanol with a yield of 14.8% at 1 : 2 ratio.

Biobutanol from lignocellulosic biomass by a novel Clostridium sporogenes BE01

In the current study, a novel non-acetone forming butanol and ethanol producer Was isolated and identified. Based on the 16s rDNA sequence BLAST and phylogenetic analyses, it was found to have high similarity with the reported hydrogen producing strains of Clostridium sporogenes. Biochemical studies revealed that it is lipase and protease positive. The lipolytic and proteolytic properties are the very important characteristics of Clostridium sporogenes. Sugar utilization profile studies were positive for glucose, saccharose, cellobiose and weakly positive result to xylose. This study demonstrated C. sporogenes BE01, an isolate from NIIST is having potential to compete with existing, well known butanol producers with the advantage of no acetone in the final solvent mixture. Rice straw hydrolysate is a potent source of substrate for butanol production by C. sporogenes BE01. Additional supplementation of vitamins and minerals were avoided by using rice straw hydrolysate as substrate. Its less growth, due to the inhibitors present in the hydrolysate and also inhibition by products resulted in less efficient conversion of sugars to butanol. Calcium carbonate played an important role in improving the butanol production, by providing the buffering action during fermentation and stimulating the electron transport mediators and redox reactions favoring butanol production. Its capability to produce acetic acid, butyric acid and hydrogen in significant quantities during butanol production adds value to the conversion process of lignocellulosic biomass to butanol. High cell density fermentation by immobilizing the cells on to ceramic particles improved the solvents and VFA production. Reduced sugar utilization from the concentrated hydrolysate could be due to accumulation of inhibitors in the hydrolysate during concentration. Two-stage fermentation was very efficient with immobilized cells and high conversions of sugars to solvents and VFAs were achieved. The information obtained from the study would be useful to develop a feasible technology for conversion of lignocellulosic biomass to biobutanol.

Projeto e construção de um bioreator para síntese orgânica assimétrica catalisada por saccharomyces cerevisiae (fermento biológico de padaria)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Modeling and simulation of cephalosporin C production in a fed-batch tower-type bioreactor

Immobilized cell utilization in tower-type bioreactor is one of the main alternatives being studied to improve the industrial bioprocess. Other alternatives for the production of beta -lactam antibiotics, such as a cephalosporin C fed-batch p recess in an aerated stirred-tank bioreactor with free cells of Cepha-losporium acremonium or a tower-type bioreactor with immobilized cells of this fungus, have proven to be more efficient than the batch profess. In the fed-batch process, it is possible to minimize the catabolite repression exerted by the rapidly utilization of carbon sources (such as glucose) in the synthesis of antibiotics by utilizing a suitable flow rate of supplementary medium. In this study, several runs for cephalosporin C production, each lasting 200 h, were conducted in a fed-batch tower-type bioreactor using different hydrolyzed sucrose concentrations, For this study's model, modifications were introduced to take into account the influence of supplementary medium flow rate. The balance equations considered the effect of oxygen limitation inside the bioparticles. In the Monod-type rate equations, eel concentrations, substrate concentrations, and dissolved oxygen were included as reactants affecting the bioreaction rate. The set of differential equations was solved by the numerical method, and the values of the parameters were estimated by the classic nonlinear regression method following Marquardt's procedure with a 95% confidence interval. The simulation results showed that the proposed model fit well with the experimental data,and based on the experimental data and the mathematical model an optimal mass flow rate to maximize the bioprocess productivity could be proposed.

Seleção de um suporte sintético para imobilizar células do Botryospaheria rhodina e comparação da produção de lacase por células livres e imobilizadas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de L-ácido lático a partir de células bacterianas imobilizadas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Utilização de Saccharomyces cerevisiae imobilizada em bagaço de cana de açúcar para a biossorção e biodegradação do corante acid black 48

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de compostos fenólicos a partir de células imobilizadas do líquen Parmotrema andinum (Müll. Arg.) Hale e avaliação de atividade antimicrobiana

A utilização de metabólitos secundários obtidos de líquens, na indústria farmacêutica, de cosmético, têxtil e de alimentos deve ser criteriosa, visto que a extração e isolamento desses metabólitos requerem uma grande quantidade de biomassa dificilmente renovável, devido ao crescimento lento do líquen. Atualmente, é possível obter substâncias liquênicas tanto por cultivo de tecidos, como por imobilizações celulares e enzimáticas, a partir do talo in natura, utilizando pequena quantidade de material liquênico. Portanto, este trabalho objetiva investigar a produção de compostos fenólicos a partir de células imobilizadas de Parmotrema andinum (Müll. Arg.) Hale utilizando acetato de sódio como precursor da biossíntese dos fenóis. Ensaios de atividade antimicrobiana com extratos orgânicos do talo in natura, eluatos celulares e do ácido lecanórico isolado de P. andinum Hale demonstraram ação contra bactérias Gram-positivas. Através de testes biocromatográficos foi possível associar a atividade antibacteriana ao ácido lecanórico e uma substância não identificada presente na espécie. As substâncias produzidas através de imobilização celular não exibiram ação inibitória frente os microrganismos testados.

Produção, imobilização e aplicação de lipase de Fusarium verticillioides

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Immobilization of marine fungi on silica gel, silica xerogel and chitosan for biocatalytic reduction of ketones

The scanning electron microscopy (SEM) analysis showed that whole living hyphal of marine fungi Aspergillus sclerotiorum CBMAI 849 and Penicillium citrinum CBMAI 1186 were immobilized on support matrices of silica gel, silica xerogel and/or chitosan. P. citrinum immobilized on chitosan catalyzed the quantitative reduction of 1-(4-methoxyphenyl)-ethanone (1) to the enantiomer (S)-1-(4-methoxyphenyl)-ethanol (3b), with excellent enantioselectivity (ee > 99%, yield = 95%). Interestingly, ketone 1 was reduced with moderate selectivity and conversion to alcohol 3b (ee = 69%, c 40%) by the free mycelium of P. citrinum. This free mycelium of P. citrinum catalyzed the production of the (R)-alcohol 3a, the antipode of the alcohol produced by the immobilized cells. P. citrinum immobilized on chitosan also catalyzed the bioreduction of 2-chloro-1-phenylethanone (2) to 2-chloro-1-phenylethanol (4a,b), but in this case without optical selectivity. These results showed that biocatalytic reduction of ketones by immobilization hyphal of marine fungi depends on the xenobiotic substrate and the support matrix used. (c) 2012 Elsevier B.V. All rights reserved.

Development of muliplexed analytical methods based on bioluminescent whole-cell biosensors

The subject of this Ph.D. research thesis is the development and application of multiplexed analytical methods based on bioluminescent whole-cell biosensors. One of the main goals of analytical chemistry is multianalyte testing in which two or more analytes are measured simultaneously in a single assay. The advantages of multianalyte testing are work simplification, high throughput, and reduction in the overall cost per test. The availability of multiplexed portable analytical systems is of particular interest for on-field analysis of clinical, environmental or food samples as well as for the drug discovery process. To allow highly sensitive and selective analysis, these devices should combine biospecific molecular recognition with ultrasensitive detection systems. To address the current need for rapid, highly sensitive and inexpensive devices for obtaining more data from each sample,genetically engineered whole-cell biosensors as biospecific recognition element were combined with ultrasensitive bioluminescence detection techniques. Genetically engineered cell-based sensing systems were obtained by introducing into bacterial, yeast or mammalian cells a vector expressing a reporter protein whose expression is controlled by regulatory proteins and promoter sequences. The regulatory protein is able to recognize the presence of the analyte (e.g., compounds with hormone-like activity, heavy metals…) and to consequently activate the expression of the reporter protein that can be readily measured and directly related to the analyte bioavailable concentration in the sample. Bioluminescence represents the ideal detection principle for miniaturized analytical devices and multiplexed assays thanks to high detectability in small sample volumes allowing an accurate signal localization and quantification. In the first chapter of this dissertation is discussed the obtainment of improved bioluminescent proteins emitting at different wavelenghts, in term of increased thermostability, enhanced emission decay kinetic and spectral resolution. The second chapter is mainly focused on the use of these proteins in the development of whole-cell based assay with improved analytical performance. In particular since the main drawback of whole-cell biosensors is the high variability of their analyte specific response mainly caused by variations in cell viability due to aspecific effects of the sample’s matrix, an additional bioluminescent reporter has been introduced to correct the analytical response thus increasing the robustness of the bioassays. The feasibility of using a combination of two or more bioluminescent proteins for obtaining biosensors with internal signal correction or for the simultaneous detection of multiple analytes has been demonstrated by developing a dual reporter yeast based biosensor for androgenic activity measurement and a triple reporter mammalian cell-based biosensor for the simultaneous monitoring of two CYP450 enzymes activation, involved in cholesterol degradation, with the use of two spectrally resolved intracellular luciferases and a secreted luciferase as a control for cells viability. In the third chapter is presented the development of a portable multianalyte detection system. In order to develop a portable system that can be used also outside the laboratory environment even by non skilled personnel, cells have been immobilized into a new biocompatible and transparent polymeric matrix within a modified clear bottom black 384 -well microtiter plate to obtain a bioluminescent cell array. The cell array was placed in contact with a portable charge-coupled device (CCD) light sensor able to localize and quantify the luminescent signal produced by different bioluminescent whole-cell biosensors. This multiplexed biosensing platform containing whole-cell biosensors was successfully used to measure the overall toxicity of a given sample as well as to obtain dose response curves for heavy metals and to detect hormonal activity in clinical samples (PCT/IB2010/050625: “Portable device based on immobilized cells for the detection of analytes.” Michelini E, Roda A, Dolci LS, Mezzanotte L, Cevenini L , 2010). At the end of the dissertation some future development steps are also discussed in order to develop a point of care (POCT) device that combine portability, minimum sample pre-treatment and highly sensitive multiplexed assays in a short assay time. In this POCT perspective, field-flow fractionation (FFF) techniques, in particular gravitational variant (GrFFF) that exploit the earth gravitational field to structure the separation, have been investigated for cells fractionation, characterization and isolation. Thanks to the simplicity of its equipment, amenable to miniaturization, the GrFFF techniques appears to be particularly suited for its implementation in POCT devices and may be used as pre-analytical integrated module to be applied directly to drive target analytes of raw samples to the modules where biospecifc recognition reactions based on ultrasensitive bioluminescence detection occurs, providing an increase in overall analytical output.

Afffinity tag excision strategies for Car9-tagged proteins

Thesis (Master's)--University of Washington, 2016-06

Mathematical Modeling for Studying Microbial Processes – Some Examples

Mathematical modeling may have different purposes in chemical and biochemical engineering sciences. One of them is to confirm or to reject kinetic models for certain processes, or to evaluate the importance of some transport phenomena on the net chemical or biochemical reaction rate. In the present paper different microbial processes are considered and modeled for evaluation of kinetic constants for batch and continuous processes accomplished by free and immobilized microbial cells. The practical examples are from the field of wastewater treatment and biosynthesis of products, like enzymes, lactic acid, gluconic acid, etc. By the aid of mathematical modeling the kinetics and the type of inhibition are specified for microbial wastewater denitrification and biodegradation of halogenated hydrocarbons. The importance of free and immobilized cells and their separate contribution to the overall microbial process is also evaluated for some fermentation processes: gluconic acid production, dichloroethane biodegradation, lactic acid fermentation and monochloroacetic acid biodegradation.

Mathematical Modeling of Perifusion Cell Culture Experiments

In perifusion cell cultures, the culture medium flows continuously through a chamber containing immobilized cells and the effluent is collected at the end. In our main applications, gonadotropin releasing hormone (GnRH) or oxytocin is introduced into the chamber as the input. They stimulate the cells to secrete luteinizing hormone (LH), which is collected in the effluent. To relate the effluent LH concentration to the cellular processes producing it, we develop and analyze a mathematical model consisting of coupled partial differential equations describing the intracellular signaling and the movement of substances in the cell chamber. We analyze three different data sets and give cellular mechanisms that explain the data. Our model indicates that two negative feedback loops, one fast and one slow, are needed to explain the data and we give their biological bases. We demonstrate that different LH outcomes in oxytocin and GnRH stimulations might originate from different receptor dynamics. We analyze the model to understand the influence of parameters, like the rate of the medium flow or the fraction collection time, on the experimental outcomes. We investigate how the rate of binding and dissociation of the input hormone to and from its receptor influence its movement down the chamber. Finally, we formulate and analyze simpler models that allow us to predict the distortion of a square pulse due to hormone-receptor interactions and to estimate parameters using perifusion data. We show that in the limit of high binding and dissociation the square pulse moves as a diffusing Gaussian and in this limit the biological parameters can be estimated.

Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.