BIOTECH 2
Microbiology, 2001 May, 147(Pt 5), 1383 - 91 Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration; Hansen MC et al.; Use of green fluorescent protein (GFP) as a molecular reporter is restricted by several environmental factors, such as its requirement for oxygen in the development of the fluorophore, and its poor fluorescence at low pH . There are conflicting data on these limitations, however, and systematic studies to assess the importance of these factors for growing bacterial cultures are lacking . In the present study, homogeneous expression of the gfpmut3* gene directed by a synthetic constitutive lactococcal promoter was demonstrated in batch cultures and in biofilms of Streptococcus gordonii DL1 . A lower limit of oxygen concentration for maturation of the GFP fluorophore was determined: fluorescence was emitted at 0.1 p.p.m . dissolved oxygen (in conventionally prepared anaerobic media lacking reducing agents), whereas no fluorescence was detected in the presence of 0.025 p.p.m . dissolved oxygen (obtained by addition of L-cysteine as reducing agent) . When an anaerobically grown (non-fluorescent) >50 microm thick biofilm was shifted to aerobic conditions, fluorescence could be detected within 4 min, reaching a maximum over the next 16 min . It was not possible to detect any fluorescence gradients (lateral or vertical) within the >50 microm thick biofilm, and fluorescence development after the shift to aerobic conditions occurred throughout the biofilm (even at the substratum) . This suggests that oxygen gradients, which might result in reduced GFP fluorescence, did not exist in the >50 microm thick biofilm of this organism . Production of lactic acid and the subsequent acidification in batch cultures of S . gordonii DL1 led to a decrease in fluorescence intensity . However, severe pH reduction was prevented when the bacterium was grown as a biofilm in a flowcell, and a homogeneous distribution of a strong fluorescence signal was observed . These findings show that GFP can be applied to studies of oxygen-tolerant anaerobic bacteria, that densely packed, flowcell-grown biofilms of S . gordonii do not develop oxygen gradients inhibitory to GFP fluorescence development, and that the often transient nature of GFP fluorescence in acid-producing bacteria can be overcome in flowcells, probably by the elimination of metabolic by-product accumulation.
Appl Environ Microbiol, 2001 May, 67(5), 2319 - 25 Shewanella putrefaciens adhesion and biofilm formation on food processing surfaces; Bagge D et al.; Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions . S . putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces . S . putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces . Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25 degrees C and reflected the cell density in suspension . Numbers of adhering bacteria from a suspension containing 10(8) CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10(2) CFU/cm(2)) than in a batch system (reaching 10(7) CFU/cm(2)), and maximum numbers were reached after 24 h . When nutrients were supplied, S . putrefaciens grew in biofilms with layers of bacteria . The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation . The number of S . putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S . putrefaciens bacteria in the biofilm . Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation . We used a combination of fluorescence microscopy (4',6'-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.
Water Res, 2001 Apr, 35(6), 1624 - 6 Helicobacter sp . recovered from drinking water biofilm sampled from a water distribution system; Park SR et al.; Workers examining the transmission route(s) and reservoir(s) of infection for Helicobacter pylori have postulated several environmental reservoirs for the organism, including water . Such work has, to date, concentrated on the bulk liquid in drinking water systems rather than on biofilms . Previous investigations by the authors have suggested biofilms in water distribution systems are a possible reservoir of infection . This current study comprised of an analysis of a section of cast iron mains distribution pipe removed from an urban environment in the north-east of Scotland during routine maintenance work . Immediately upon removal of the pipe section, the interior lumen was swabbed to remove the biofilm layer . Subsequent analysis for the presence of Helicobacter DNA using a nested PCR approach produced a positive result . This data provides the first evidence for the existence of Helicobacter in biofilms found in water distribution systems anywhere in the world.
Water Res, 2001 Apr, 35(6), 1379 - 86 Methane microprofiles in a sewage biofilm determined with a microscale biosensor; Damgaard LR et al.; Microprofiles of the methane concentration in a 3.5-mm-thick sewage outlet biofilm were measured at high spatial and temporal resolution using a microscale biosensor for methane . In the freshly collected biofilm, methane was building up to a concentration of 175 mumol l-1 at 3 mm depth with a total methanogenesis of 0.14 mumol m-2 s-1, as compared to an aerobic respiration (including methane oxidation) of 0.80 mumol m-2 s-1 . A model biofilm was established by homogenisation of an in situ biofilm and 12 days of incubation with surplus sodium acetate . The homogenised biofilm was able to maintain 50% of the methanogenic activity in the absence of external electron donor . Oxygen had only a minor effect on the methane production, but aerobic respiration consumed a substantial part of the produced methane and was thus an important control on methane export from the biofilm . A concentration of 2 mmol l-1 nitrate was shown to inhibit methanogenesis only in the upper layer of the biofilm, whereas a further addition of 2 mmol l-1 sulphate inhibited methanogenesis in the entire biofilm . The study demonstrated the power of the methane microsensor in the study of microhabitats with concurrent production and consumption of methane.
J Clin Pediatr Dent, 2000 Spring, 24(3), 237 - 43 Effects of three different infant dentifrices on biofilms and oral microorganisms; Modesto A et al.; The purpose of this work was to evaluate the effects of infant dentifrices: A--with lactoperoxidase, glucose oxidase and lactoferrin; B--with 1100 ppm of NaF and sodium lauryl sulfate; C--with extract of calendula . The dentifrices were test on biofilms formed in vitro from saliva and dental plaque of infants, using reference strains A . viscosus (ATCC 43146); C . albicans (ATCC 51501); L . casei (ATCC 4646); S . mitis (ATCC 49456); S . mutans (ATCC 25175); S . oralis (ATCC 35037); S . sanguis (ATCC 10586); S . sobrinus (ATCC 27609) and isolated clinically microorganisms C . albicans, S . mitis, S . mutans, S . oralis, S . sanguis, S . sobrinus and Lactobacillus sp . Twenty infants were chosen, who were beginning treatment at the Infants Clinic of the Pediatric Dentistry Department, Federal University of Rio de Janeiro . A pool of unstimulated saliva and a pool of dental plaque were collected from which biofilms were produced . Supernatants from each dentifrice were prepared and concentrated and diluted solutions of the dentifrices and a control sterile diluent were tested against the biofilms produced, for 1 and 3 minutes, and against the microorganisms . The results were statistically analyzed by the ANOVA and Tukey Test . After the exposure of the biofilms produced both from saliva and from dental plaque, to the dentifrice B concentrated and 1/2, for 1 and 3 minutes, the viable microorganisms count (CFU/ml), compared to the controls, was significantly reduced (p < 0.05) . However, exposure to the dentifrices A and C concentrated and dentifrice B 1/4 and 1/8, for 1 and 3 minutes, was not significantly lethal to the biofilms . The dentifrices A and C, either concentrated or diluted (1/2 to 1/128) and the dentifrice B in the dilutions 1/16 to 1/128 did not have an antimicrobial effect on any microorganism evaluated . For all the microorganisms evaluated, the dentifrice B concentrated and in the 1/2 dilution showed a significant antimicrobial effect, when compared with the control (p < 0.05).
FEMS Microbiol Lett, 2001 Apr 13, 197(2), 151 - 7 Characterization and expression of secA in Mycobacterium avium; Limia A et al.; Mycobacterium avium is both a pathogen that infects several hosts such as humans, pigs, and birds, as well as a microorganism that is encountered in environmental sources (soil and water) . Protein secretion by the bacterium is likely to influence its ability to overcome adverse and competitive conditions both within or outside the host . Using a combination of cloning and information available in the databank, we characterized the secA gene from M . avium, encoding for a major preprotein translocase subunit associated with the secretion system of prokaryotics . In addition, we cloned the secA promoter sequence in a reporter construct upstream of a promoterless gfp . It was determined that the secA of M . avium shares large homology with the secA of Mycobacterium tuberculosis but not with secA of Mycobacterium leprae . secA expression was determined to be greater at logarithmic growth phase although it was also expressed at low levels during the stationary phase . secA expression was also observed when the bacteria were incubated in water as well as within human monocyte-derived macrophages and in conditions that are associated with biofilm formation . Future evaluation of the sec pathway in M . avium might provide important information about secreted proteins that are required for survival in different environments.
Ann N Y Acad Sci, 1999 Jun 18, 875, 84 - 104 Physico-chemical and mass transfer considerations in microencapsulation; Goosen MF; To gain better insight into mass transfer problems in encapsulated cell systems requires a combination of experimental investigations and mathematical modeling . Specific mass transfer studies are reviewed including oxygen transfer in immobilized animal cell culture bioreactors, modeling of polymer droplet formation and encapsulated animal cell growth, and growth of somatic tissue encapsulated in alginate using electrostatics . Special emphasis is given to electrostatic droplet generation for cell immobilization.
Can J Microbiol, 1999 Mar, 45(3), 235 - 41 Removal of phenolic compounds from a petrochemical effluent with a methanogenic consortium; Charest A et al.; A methanogenic consortium was used to degrade phenol and ortho- (o-) cresol from a specific effluent of a petrochemical refinery . This effluent did not meet the local environmental regulations for phenolic compounds (178 mg/L), oils and greases (61 mg/L), ammoniacal nitrogen (75 mg/L) or sulfides (3.2 mg/L) . The consortium, which degrades phenol via its carboxylation to benzoic acid, was progressively adapted to the effluent . Despite the very high effluent toxicity (EC50 of 2% with Microtox), the adapted consortium degraded 97% of 156 mg/L phenol in the supplemented effluent after 13 days in batch cultures (serum bottle) . The addition of proteose peptone to the effluent is essential for phenol degradation . o-cresol was also transformed but not meta- or para-cresols . A continuous flow fixed-film anaerobic bioreactor was developed with the consortium . Treating the effluent with the bioreactor reduced phenol and phenolic compounds concentrations by 97 and 83%, respectively, for a hydraulic residence time of 6 h . This treatment also reduced by about half the effluent toxicity . Oils and greases and ammoniacal nitrogen were not affected . Similar microbiological forms were observed in serum bottles and in the bioreactors with or without the petrochemical effluent . These results indicate that this methanogenic consortium can treat efficiently the phenolic compounds in this specific petrochemical effluent.
J Immunother, 1999 Jul, 22(4), 299 - 307 Large-scale production of natural cytokines during activation and expansion of human T lymphocytes in hollow fiber bioreactor cultures; Lamers CH et al.; We studied the large-scale production of a variety of natural cytokines during the activation and expansion of human T lymphocytes in a hollow fiber bioreactor culture system . Peripheral blood mononuclear cells (PBMC) were activated using phytohemagglutinin plus recombinant interleukin-2 (IL-2) . Phytohemagglutinin was either present in the hollow fiber bioreactor during the entire 15-16-day culture period or only during the 20-h preactivation of the PBMC in culture bags . The expanding T lymphocytes were mainly CD3+,8+ and exerted maximal natural, activated, bispecific monoclonal antibody-redirected and lectin-dependent cytolytic activities between days 9 and 13 of culture . IL-1 and IL-4 were only produced in low amounts . IL-8 and lymphotoxin were primarily produced during the first week of culture . Harvest of the hollow fiber bioreactor culture supernatant at the time of peak cytokine concentration would have yielded per 10(8) PBMC input between 3.7 and 4.9 micrograms of IL-8 (at days 2 or 3), and between 0.02 and 0.5 microgram of lymphotoxin (at days 6 or 7) . Tumor necrosis factor-alpha and IL-6 were produced during the entire culture period of 15 or 16 days: per 10(8) PBMC input, between 0.1 and 0.4 microgram of tumor necrosis factor-alpha (at days 2 or 3) and between 0.03 and 0.5 microgram of IL-6 (at days 15 or 16) . Production of interferon-gamma and granulocyte-macrophage colony-stimulating factor started from initiation of cultures onwards to reach peak levels at the end of the 15- or 16-day culture period, yielding at that time between 2.1 and 17.7 micrograms/ml of interferon-gamma and between 0.4 and 4.2 micrograms of granulocyte-macrophage colony-stimulating factor per 10(8) PBMC input . The production of tumor necrosis factor-alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor was proportional to the extent of lymphocyte multiplication . These results demonstrate the usefulness of hollow fiber bioreactor cultures to produce natural cytokines during the activation and expansion of predominantly CD3+,8+ T lymphocytes.
Biotechnol Bioeng, 1999 Sep 5, 64(5), 580 - 9 Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization; Carrier RL et al.; Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair . We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors . In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle . Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)) . Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05) . Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape . Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks . After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue .
Appl Biochem Biotechnol, 1999 Spring, 77-79, 455 - 71 Bioconversion of mixed solids waste to ethanol; Nguyen QA et al.; A mixed solids waste (MSW) feedstock, comprising construction lumber waste (35% oven-dry basis), almond tree prunings (20%), wheat straw (20%), office waste paper (12.5%), and newsprint (12.5%), was converted to ethanol via dilute-acid pretreatment followed by enzymatic hydrolysis and yeast fermentation . The MSW was pretreated with dilute sulfuric acid (0.4% w/w) at 210 degrees C for 3 min in a 4-L steam explosion reactor, then washed with water to recover the solubilized hemicellulose . The digestibility of water-washed, pretreated MSW was 90% in batch enzymatic hydrolysis at 66 FPU/g cellulose . Using an enzyme-recycle bioreactor system, greater than 90% cellulose hydrolysis was achieved at a net enzyme loading of about 10 FPU/g cellulose . Enzyme recycling using membrane filtration and a fed-batch fermentation technique is a promising option for significantly reducing the cost of enzyme in cellulose hydrolysis . The hexose sugars were readily fermentable using a Saccharomyces cerevisiae yeast strain that was adapted to the hydrolysate . Solid residue after enzyme digestion was subjected to various furnace experiments designed to assess the fouling and slagging characteristics . Results of these analyses suggest the residue to be of a low to moderate slagging and fouling type if burned by itself.
Biotechnol Bioeng, 1999 Aug 20, 64(4), 459 - 77 Integrated two-liquid phase bioconversion and product-recovery processes for the oxidation of alkanes: process design and economic evaluation Mathys RG, Schmid A, Witholt B. Pseudomonas oleovorans and recombinant strains containing the alkane oxidation genes can produce alkane oxidation products in two-liquid phase bioreactor systems . In these bioprocesses the cells, which grow in the aqueous phase, oxidize apolar, non-water soluble substrates . The apolar products typically accumulate in the emulsified apolar phase . We have studied both the bioconversion systems and several downstream processing systems to separate and purify alkanols from these two-liquid phase media . Based on the information generated in these studies, we have now designed bioconversion and downstream processing systems for the production of 1-alkanols from n-alkanes on a 10 kiloton/yr scale, taking the conversion of n-octane to 1-octanol as a model system . Here, we describe overall designs of fed-batch and continuous-fermentation processes for the oxidation of octane to 1-octanol by Pseudomonas oleovorans, and we discuss the economics of these processes . In both systems the two-liquid phase system consists of an apolar phase with hexadecene as the apolar carrier solvent into which n-octane is dissolved, while the cells are present in the aqueous phase . In one system, multiple-batch fermentations are followed by continuous processing of the product from the separated apolar phase . The second system is based on alkane oxidation by continuously growing cultures, again followed by continuous processing of the product . Fewer fermentors were required and a higher space-time-yield was possible for production of 1-octanol in a continuous process . The overall performance of each of these two systems has been modeled with Aspen software . Investment and operating costs were estimated with input from equipment manufacturers and bulk-material suppliers . Based on this study, the production cost of 1-octanol is about 7 US$kg-1 when produced in the fed-batch process, and 8 US$kg-1 when produced continuously . The comparison of upstream and downstream capital costs and production costs showed significantly higher upstream costs for the fed-batch process and slightly higher upstream costs for continuous fermentation . The largest cost contribution was due to variable production costs, mainly resulting from media costs . The organisms used in these systems are P . putida alk+ recombinants which oxidize alkanes, but cannot oxidize the resulting alkanols further . Hence, such cells need a second carbon source, which in these systems is glucose . Although the continuous process is about 10% more expensive than the fed-batch process, improvements to reduce overall cost can be achieved more easily for continuous than for fed-batch fermentation by decreasing the dilution rate while maintaining near constant productivity . Improvements relevant to both processes can be achieved by increasing the biocatalyst performance, which results in improved overall efficiency, decreased capital investment, and hence, decreased production cost .
Biotechnol Bioeng, 1999 Aug 20, 64(4), 452 - 8 Membrane sparger in bubble column, airlift, and combined membrane-ring sparger bioreactors Poulsen BR, Iversen JJ. The bubble column and the two internal loop airlift reactors (riser/downcomer area ratios of 0.11 and 0.58) characterized in this study were equipped with a rubber membrane sparger, which produced small bubbles, giving high mass transfer coefficients . The low mixing intensity in the bubble column was increased by an order of magnitude in the airlift reactors . We designed a novel aeration and mixing system by adding a ring sparger to the membrane sparger in the bubble column and maintained the advantages of both airlift configuration (good mixing properties) and bubble column configuration (efficient aeration, without any internal constructions) . The combined membrane-ring sparger system has unique features with respect to the efficiency of utilization of substrate gasses and energy . Model experiments showed that the small bubbles from the membrane sparger do not coalesce with the large bubbles from the ring sparger . If different gases were added through the two spargers it was possible to transfer a hazardous or expensive gas quantitatively to the liquid through the membrane sparger (dual sparging mode) . In the combined membrane-ring sparger system the energy input for mixing and mass transfer is divided . Therefore, the energy consumption can be minimized if the flow distribution of air through the membrane and ring sparger is controlled by the oxygen demand and the inhomogeneity of the culture, respectively (split sparging mode) . The dual sparging mode was used for mass production of the alga Rhodomonas sp . as the first step in aquatic food chains . Avoiding mechanical parts removes an important risk of malfunction, and a continuous culture could be maintained for more than 8 months .
Biotechnol Bioeng, 1999 Jul 5, 64(1), 14 - 26 Characterization of bimodal cell death of insect cells in a rotating-wall vessel and shaker flask Cowger NL, O'Connor KC, Hammond TG, Lacks DJ, Navar GL. In previous publications, we reported the benefits of a high-aspect rotating-wall vessel (HARV) over conventional bioreactors for insect-cell cultivation in terms of reduced medium requirements and enhanced longevity . To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker-flask control . Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period . We present a kinetic model of growth and bimodal cell death . The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways . Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis . The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions . In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first-order term . Cell-cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels . For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 +/- 1% to 24 +/- 4% . The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures . Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell-cell interactions, waste accumulation, and mass transport . These studies may benefit insect-cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity .
Biotechnol Bioeng, 1999 Jun 5, 63(5), 601 - 11 Epoxidation of 1,7-octadiene by pseudomonas oleovorans in a membrane bioreactor Doig SD, Boam AT, Livingston AG, Stuckey DC. A growing cell culture of Pseudomonas oleovorans was used to biotransform 1,7-octadiene to 1,2-epoxy-7,8-octene in a continuous-flow bioreactor with an external membrane module . A dense silicone rubber membrane was used to contact an organic phase, containing both the reactant (1,7-octadiene) and the growth substrate (heptane), with an aqueous biomedium phase containing the biocatalyst . Heptane and octadiene delivery to the aqueous phase, and epoxide extraction into the solvent, occurred by diffusion across the dense membrane under a concentration-driving force . In addition, a liquid feed of heptane and octadiene was pumped directly into the bioreactor to increase the rate of delivery of these compounds to the aqueous phase . In this system 1,2-epoxy-7,8-octene accumulated in a pure solvent phase, thus, product recovery problems associated with emulsion formation were avoided . Furthermore, no phase breakthrough of either liquid across the membrane was observed . In this system, the highest volumetric productivity obtained was 30 U.L-1, and this was achieved at a dilution rate of 0.07 h-1, 70 m2 . m-3 of membrane area, and a steady-state biomass concentration of 2 . 5 g.L-1 . The system was stable for over 1250 h . Decreasing the dilution rate led to an increased biomass concentration, however, the specific activity was significantly reduced, and therefore, an optimal dilution rate was determined at 0.055 h-1 .
Biotechnol Bioeng, 1999 Jun 5, 63(5), 593 - 600 Studies on the respiration rate of free and immobilized cells of cephalosporium acremonium in cephalosporin C production Araujo ML, Giordano RC, Hokka CO. Bioprocesses using filamentous fungi immobilized in inert supports present many advantages when compared to conventional free cell processes . However, assessment of the real advantages of the unconventional process demands a rigorous study of the limitations to diffusional mass transfer of the reagents, especially concerning oxygen . In this work, a comparative study was carried out on the cephalosporin C production process in defined medium containing glucose and sucrose as main carbon and energy sources, by free and immobilized cells of Cephalosporium acremonium ATCC 48272 in calcium alginate gel beads containing alumina . The effective diffusivity of oxygen through the gel beads and the effectiveness factors related to the respiration rate of the microorganism were determined experimentally . By applying Monod kinetics, the respiration kinetics parameters were experimentally determined in independent experiments in a complete production medium . The effectiveness factor experimental values presented good agreement with the theoretical values of the approximated zero-order effectiveness factor, considering the dead core model . Furthermore, experimental results obtained with immobilized cells in a 1.7-L tower bioreactor were compared with those obtained in 5-L conventional fermentor with free cells . It could be concluded that it is possible to attain rather high production rates working with relatively large diameter gel beads (ca . 2.5 mm) and sucrose consumption-based productivity was remarkably higher with immobilized cells, i.e., 0.33 gCPC/kg sucrose/h against 0.24 gCPC/kg sucrose/h in the aerated stirred tank bioreactor process .
Biotechnol Bioeng, 1999 Aug 20, 64(4), 401 - 17 Incorporation of ammonium into intracellular UDP-activated N-acetylhexosamines and into carbohydrate structures in glycoproteins; Valley U et al.; The negative effects of ammonia on animal cells, especially in vitro cultures, are well known, but the mechanism of how ammonia inhibits cell growth and influences the glycosylation of proteins is not completely understood . We investigated the ammonium action on the synthesis of the intracellular UDP-N-acetylhexos- amines (UDPGNAc), which are precursors of glycosylation as well as on N-linked oligosaccharides of a recombinant human IL-2 mutant variant model glycoprotein expressed in BHK-21 cells under defined and controlled culture conditions in a continuously perfused bioreactor . The examinations were based on our previous observations that increased ammonia concentrations in the medium lead to the intracellular formation and accumulation of UDPGNAc (Ryll et al., 1994) . The kinetics of formation of the UDPGNAc pool after adding ammonia and its reconstitution to normal conditions are shown . To study the pathway leading to the intracellular increase of UDPGNAc, the uptake and incorporation of 15NH4+ was confirmed by the detection of 15N in UDP-N-acetylglucosamine (UDP-GlcNAc) . UDP-GlcNAc was purified using high pH anion-exchange chromatography with pulsed amperometric detection and analyzed by GC/MS . The proportion of UDP-GlcNAc containing 15N was approximately 60% and corresponds quantitatively to the increased intracellular concentration of UDP-GlcNAc . In order to confirm the direct influence of ammonia on protein glycosylation, the human IL-2 mutant glycoprotein variant IL-Mu6, bearing a novel N-glycosylation site, has been produced under defined protein-free medium conditions in the presence of 15NH4Cl . IL-Mu6 glycoprotein was purified and N-glycans released were analyzed by matrix-assisted laser desorption ionization time of flight mass spectroscopy . Maximally 60-80% of N-acetylated sugars in N-glycan structures contained 15N indicating that ammonium is used as a building block during synthesis of the carbohydrate structures expressed from in vitro cultivated mammalian cells .
Biotechnol Bioeng, 1999 Aug 5, 64(3), 357 - 67 Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes; Grammatikos SI et al.; Efficient cell culture process development for the industrial production of recombinant therapeutics is characterized by constraints which pertain to issues such as costs, competitiveness and the meeting of project timelines . These constraints require tools which can help the developer learn as much as possible as quickly as possible about the cell at hand and identify features of a particular culture which are amenable to improvement . Current on- and off-line monitoring parameters, however useful, provide only late indications (cell concentration, viability) and circumstantial evidence (lactate, ammonia, etc.) with regard to the physiologic status of cells at the time of sampling . The relative intracellular content of purine to pyrimidine nucleotide triphosphates as well as the ratio of UTP to UDP-N-acetylhexosamines have been previously described as sensitive indicators of a cell's metabolic status, growth potential, and overall physiological condition . The sensitivity of such nucleotide ratios and their usefulness in commercially relevant process development and characterization were tested at Boehringer Ingelheim Pharma KG in a large number of fermentations (>80) with a variety of culture modes, cells, and products in scales up to 10,000 litres . Monitoring of these intracellular parameters allows a timely and reliable assessment of cell state and growth potential, which is possible neither by classical cell number and viability measurements nor by a variety of fermentation data typically monitored . The view inside the cell afforded by nucleotide monitoring enables prediction of the behavior of a culture up to 2 days before any hint of physiological changes is given by cell number and viability estimation . In this paper, data relating the growth behavior of CHO and hybridoma cell lines to their nucleotide pools are shown . Two very different processes for the production of recombinant tPA in 10,000-litre bioreactors are compared and characterized with respect to their nucleotide profiles . Examples from industrial process development cases in which intracellular nucleotide information is used to advantage are also presented and discussed .
Biotechnol Bioeng, 1999 Jul 20, 64(2), 194 - 9 Efficient coupled transcription/translation from PCR template by a hollow-fiber membrane bioreactor; Nakano H et al.; A novel bioreactor using a hollow-fiber membrane was developed for the coupled transcription/translation system using T7 RNA polymerase and Escherichia coli S30 extract . The large surface area per the reaction volume of the reactor assured rapid mass transfers of substrates into the reaction mixture and of wastes out from it across the membrane by their molecular diffusion . The flux was large enough to maintain nucleotide concentrations for more than 3 h, which increased the protein synthesis greatly . In addition, the T7 terminator sequence downstream from the reporter genes was found to increase the synthesized protein significantly, especially when the product of polymerase chain reaction (PCR) was used as a template . Implementation of this finding and use of the bioreactor developed multiplied the productivity of protein by the in vitro direct expression from PCR template .
Biotechnol Bioeng, 1999 Jun 5, 63(5), 618 - 24 Regional heparinization via simultaneous separation and reaction in a novel Taylor-Couette flow device; Ameer GA et al.; The development of a safe and efficient bioreactor design has remained a challenge for the clinical application of immobilized enzymes . Specifically, the use of immobilized heparinase I has been the target of many studies to make heparin anticoagulation therapy safer for the critically ill patient with kidney failure or heart disease . We have investigated the use of Taylor-Couette flow for a novel type of bioreactor . In a previous study, we showed that the fluidization of agarose immobilized heparinase within Taylor vortices in whole blood can lead to extensive blood damage in the form of cell depletion and hemolysis . Based on these findings, we designed and developed a reactor, referred to as vortex-flow plasmapheretic reactor (VFPR), that incorporated plasmapheresis and fluidization of the agarose in the reactive compartment, separate from the whole-blood path . In the present study, immobilized heparinase I was tested as a means of achieving regional heparinization of a closed circuit . This is a method in which heparin is infused into the extracorporeal circuit predialyzer and neutralized postdialyzer . Saline studies were performed with an immobilized heparinase I-packed bed and with the VFPR . An in vitro feasibility study was performed with the VFPR using human blood . The VFPR achieved heparin conversions of 44 +/- 0.5% and 34 +/- 2% in saline and blood, respectively . In addition, the VFPR caused no blood damage . We report a novel method to achieve fluidization which depended on secondary, circumferencial flow, and was independent of the primary flow through the device .
Compost is a common name for humus, which is the result of the decomposition of organic matter. Decomposition is performed primarily by microbes, although larger creatures such as worms and ants contribute to the process. Decomposition occurs naturally in all but the most hostile environments, such as buried in landfills or in extremely arid deserts, which prevent the microbes and other decomposers from thriving.
Composting is the controlled decomposition of organic matter. Rather than allowing nature to take its slow course, a composter provides an optimal environment in which decomposers can thrive. To encourage the most active microbes, the compost pile needs the proper mix of the following ingredients: Carbon Nitrogen Oxygen (air) Water. Decomposition happens even in the absence of some of these ingredients, but not nearly as quickly and not nearly as pleasantly (for example, the plastic bag of vegetables in your refrigerator is decomposed by microbes, but the absence of air encourages anaerobic microbes that produce disagreeable odors).
All guidelines for building compost piles have the goal of creating the proper environment for a decomposing ecosystem. The ecosystem in a compost pile is a microcosm of larger ecosystems. The correct environment must be maintained for a healthy and vigorous community of decomposers. In addition to the decomposers that work directly on the organic content of the pile, compost piles provide habitat for those that prey upon direct decomposers. Their waste also becomes part of the process.
The most effective decomposers are bacteria and other microorganisms. Also important are fungi, molds, protozoa, and actinomycetes--which is something between a fungus and a mold and is often seen as white filaments in decomposing organic matter. At a macroscopic level, earthworms, ants, snails, slugs, millipedes, sow bugs, springtails, and others work on consuming and breaking down the organic matter. Centipedes and other predators feed upon these decomposers.
Compost ingredients The goal in a compost pile is to provide a healthy environment--and nutrition--for the rapid decomposers, the bacteria.
The most rapid composting occurs with the ideal ratio--by dry chemical weight--of carbon to nitrogen, from 25-to-1 to 30-to-1. In other words, the ingredients placed in the pile should contain 30 times as much carbon as nitrogen. For example, grass clippings average about 19-to-1 and dry autumn leaves average about 55-to-1. Mixing equal parts by volume approximates the ideal range. Commercial-grade composting operations pay strict attention to this ratio. For backyard composters, however, the charts of carbon and nitrogen ratios in various ingredients and the calculations required to get the ideal mixture can be intimidating, so many rules of thumb exist to guide composters in approximating this mixture.
High-carbon sources provide the cellulose needed by the composting bacteria for conversion to sugars and heat.
High-nitrogen sources provide the most concentrated protein, which allow the compost bacteria to thrive.
Some ingredients with higher carbon content:
Dry, straw-type material, such as cereal straws Autumn leaves Sawdust and wood chips Some paper and cardboard (such as corrugated cardboard or newsprint with soy-based inks) Some ingredients with higher nitrogen content:
Wilted green material (usually crop residues, or plants mowed for the purpose) Animal manures (vegetarians, not meat-eaters) Grass clippings Fruit and vegetable trimmings, skins, and waste Poultry manure provides lots of nitrogen but little carbon. Horse manure provides both. Sheep and cattle manure don't drive the compost heap to as high a temperature as poultry or horse manure, so the heap takes longer to produce the finished product.
In an attempt to judge the proper mix of materials, different rules of thumb are available. Some prefer to add one basket full of nitrogen source followed by one basket of carbon source. Mixing the materials as they are added increases the rate of decomposition, but some people prefer to place the materials in alternating layers, approximately 15 cm (6 inches) thick, to help estimate the quantities. Keeping carbon and nitrogen sources separated in the pile can slow down the process but decomposition will occur in any event.
Composting techniques There are two primary methods of aerobic composting:
Active (or hot) composting, which allows the most effective decomposing bacteria to thrive, kills most pathogens and seeds, and rapidly produces usable compost Passive (or cold) composting, which lets nature take its course in a more leisurely manner and leaves many pathogens and seeds dormant in the pile Most commercial and industrial composting operations use active composting techniques. This ensures a higher quality product and produces results in the shortest time (SEE compost windrow turner).
Home composters use a range of techniques varying from extremely passive composting (throw everything in a pile in a corner and leave it alone for a year or two) to extremely active (monitoring the temperature, turning the pile regularly, and adjusting the ingredients over time) and combinations of both.
Some composters use mineral powders to absorb smells, although a well-maintained pile seldom has bad odors.
Microbes and heating the pile An effective compost pile is kept about as damp as a well wrung-out sponge. This provides the moisture that all life needs to survive; in a compost pile, it provides an environment in which microbes can begin to do their work. Bacteria and other microorganisms fall into a variety of groups in terms of what their ideal temperature is and how much heat they generate as they do their work. Mesophilic bacteria enjoy midrange temperatures, from about 20 to 40 °C (70 to 110 °F). As they decompose the organic matter, they generate heat, and the inner part of a compost pile heats up the most.
The heap should be about 1 m (3 ft) wide, 1 m (3 ft) tall, and as long as is practicable – the advantage to making the heap at least 1 m³ (1 yd³) is that it provides suitable insulating mass to allow a good heat build-up as the material decays. The ideal temperature range hovers around 60 °C (140 °F), which kills most pathogens and weed seeds and also provides a suitable environment for thermophilic (heat-loving) bacteria, which are the fastest acting decomposers. The centre of the heap should get quite warm, possibly hot enough to burn a bare hand. If this fails to happen, common reasons include the following:
The heap is too wet, thus excluding the oxygen required by the compost bacteria The heap is too dry, so that the bacteria do not have the moisture needed to survive and reproduce There is insufficient protein (nitrogen rich material) The solution is to add material, if necessary, and/or to turn the pile to aerate it.
Depending on how quickly the compost is required, the heap can be turned one or more times to bring the outer layers to the inside of the heap and vice versa, as well as to aerate the mixture. Adding water at this time keeps the pile as damp as a wrung-out sponge. One guideline is to turn the pile when the high temperature has begun to drop, indicating that the food source for the fastest-acting bacteria (in the center of the pile) has been largely consumed. After the temperature stops rising after the pile has been turned, there is no further advantage in turning the pile. When all the material has become barely recognisable from the original ingredients, turning into dark brown or nearly black crumbly matter, it's ready to use. Some practitioners like to leave the compost to mature further for up to a year as this seems to make the benefits of compost last longer.
Other ingredients Some like to put special materials and activators into their compost. A light dusting of agricultural lime (not on the animal manure layers) can curb excessive acidity that can slow down the fermentation. Seaweed meal can provide a ready source of trace elements. Finely pulverised rock dust can also provide needed minerals, but watch out for rock dust that consists mostly of clay.
The animal manure part of compost source materials can be collected by composting toilets (in this case, human feces). However, such compost is usually not used as a fertilizer for plants that are directly edible (e.g., salad crops) but should instead be used on trees, bush fruits or else on the ornamental garden.
Bioaugmentation refers to the introduction of a group of natural microbial strain or a genetically engineered variant so as to achive bioremediation.
Usually the step involves studying the indigenous varieties present in the location. If the indigenous variety do not have the metabolic machinery that can do the remediation process, exogenous varieties with such sophisticated pathways are introduced.
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