Microfiltration cell-recycle pilot system for continuous thermoanaerobic production of exo-beta-amylase

A microfiltration cell-recycle pilot-scale system was developed comprised of a conventional continuous-flow fermentor connected to an in situ steam-sterilizable cross-flow ceramic filter with a backflushing device. A microcomputer was used to control filtration pressure, tangential flow velocity, and backflushing. Performance of the system was tested with the anaerobic production of thermostable extracellular beta-amylase at 60 degrees C by Clostridium thermosulfurogenes on maltose or malto-dextrin media. Filtration rates during continuous cultivation were between 20 and 60 L/m(2)/h. The maltodextrin and cell debris occurring at high retentate flow rates or filtration pressures impaired the performance of the filter. Backflushing initially improved the permeate flux to 42% in a maltose medium and to 10% in a maltodextrin medium, but the effect diminished with time. The productivity of beta-amylase (as much as 48 U/mL/h) and concentration of biomass (as much as 14 g/L) were increased 11- and 12-fold, respectively, if compared to values obtained in a chemostat. The concentration of beta-amylase rose to 220 U/mL in the reactor, which was 5.5-fold more than under comparable conditions in a chemostat.     

Filtration-based perfusion of hybridoma cultures in protein-free medium: Reduction of membrane fouling by medium supplementation with DNase I

In this study, a filtration-based perfusion process was developed for the production of monoclonal antibodies (IgM) by suspended hybridoma cells grown in protein-free medium. It was found that the use of protein-free medium for perfusion culture generated the formation of numerous visible suspended particles consisting of dead cells and cellular debris aggregated into fibrous material. Surprisingly high apparent viabilities were observed in such protein-free cultures. In addition, membrane fouling occurred more rapidly in protein-free medium than in conventional serum-supplemented medium. By the addition of deoxyribonuclease I (DNase I) to the protein-free medium, it was possible to prevent the formation of aggregates and to follow the evolution of the total cell population more accurately. Moreover, DNase I significantly reduced the fouling of filtration membranes, and that, for two different types of separation systems (cross-flow and vortex-flow filtration) and two different types of membranes (polycarbonate and hydrophilized polysultone). From these results, it is clear that the presence of DNA fragments liberated following cellular death is playing an important role in membrane fouling. Longevity of filtration membranes was found to be considerably greater using a vortex-flow filtration module than with a static plate-and-frame cross-flow filtration module. The use of vortex-flow filtration of conjuction with DNase I allowed maintenance of perfusion cultures for more than 1 month without membrane fouling or antibody retention and with a constant permeate IgM concentration of 250 mg/L. Hybridomacells appeared to gradually adapt to increasing rotational speed in the vortex-flow filtration module.     

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.     

Production and separation of alpha-agarase from Altermonas agarlyticus strain GJ1B

This paper presents results on the production of alpha-agarase by a fermentation process and its separation using membrane microfiltration (MF). Optimization of fermentation conditions for alpha-agarase production using Altermonas agarlyticus grown on medium containing agar as a carbon source was done in batch, fed-batch and continuous fermentations. Continuous culture at a dilution rate of 0.03 h(-1) appeared to be best suited for production of alpha-agarase by this organism. At 0.03 h(-1) dilution rate, enzyme activity was 0.9 U/ml. Clarification of broth was done using a hollow-fibre microfiltration membrane. The influence of hydrodynamic parameters on permeate flux and enzyme activity was studied. The best performance was obtained with prefiltered fermentation broth. A stable permeate flux of about 250-270 ml/min.m2 and an enzyme retention rate between 0% and 25% was obtained at temperatures between 6 degrees C and 22 degrees C, transmembrane pressure of 100 mm Hg and fluid cross-flow velocity of 4 x 10(-2) m/s. From the experiments on concentration of fermentation broth, the best compromise between enzyme activity transmission and permeate flux was obtained at a concentration factor of 2.     

Cross-flow membrane microfiltration of a bacteriol fermentation broth

Although cross-flow membrane filtration is a very attractive option for harvesting cells and recovering enzymes from cell homogenates, the process is not without its problems. Foremost of these is the deposit of dissolved and suspended solutes onto the membrane surface during operation. The formation of these dense and sometimes compressive sublayers (often called cakes) offers additional resistance to axial and permeate flows and often affects the retention characteristics of the process. In view of the complex nature of the sublayer formation process and its sensitivity to cross-flow velocity, this investigation was undertaken to determine the main factors responsible for the decline in performance during the harvesting of B. polymyxa broth by membrane microfiltration. System parameters varied include axial flow rate, concentration of cells, proteins and other components in the feed, membrane materials (ceramic, polypropylene, and stainless steel), and cleaning methods. To help explain the observed results, a new mass transport model-the solids flux model-based on the assumptions that back migration of particles from the sublayer or membrane surface is negligible and that particles that reach the solid-solution interface attach (stick) completely, is tested. Using a variety of diagnostic methods, magnesium ammonium phosphate precipitate is formed during steam sterilization of the medium and is implicated as the major foulant in this study.     

Effect of Permeate Drag Force on the Development of a Biofouling Layer in a Pressure-Driven Membrane Separation System

The effect of permeate flux on the development of a biofouling layer on cross-flow separation membranes was studied by using a bench-scale system consisting of two replicate 100-molecular-weight-cutoff tubular ultrafiltration membrane modules, one that allowed flow of permeate and one that did not (control). The system was inoculated with Pseudomonas putida S-12 tagged with a red fluorescent protein and was operated using a laminar flow regimen under sterile conditions with a constant feed of diluted (1:75) Luria-Bertani medium. Biofilm development was studied by using field emission scanning electron microscopy and confocal scanning laser microscopy and was subsequently quantified by image analysis, as well as by determining live counts and by permeate flux monitoring. Biofilm development was highly enhanced in the presence of permeate flow, which resulted in the buildup of complex three-dimensional structures on the membrane. Bacterial transport toward the membrane by permeate drag was found to be a mechanism by which cross-flow filtration contributes to the buildup of a biofouling layer that was more dominant than transport of nutrients. Cellular viability was found to be not essential for transport and adhesion under cross-flow conditions, since the permeate drag overcame the effect of bacterial motility.     

Viability of an ectomycorrhizal fungus during cross-flow filtration

Factors affecting the viability and infectivity of an ectomycorrhizal fungus during moderate concentration by cross-flow filtration were determined. Mycelial suspensions were concentrated with three commercial membrane filters (Prostak Millipore Co., M14 Tech-Sep Co. and Ceraflo Norton Co.) under aseptic conditions. Medium components may reduce the filtration rate due to their low solubility. An antifoam agent did not reduce the average flux rates as much as did the malt extract. Clear unobstructed channels (I.D. 6mm) of the tubular modules (Tech-Sep) gave the best results both in terms of performance (filtration rate) and cell viability. Shear stresses caused by pumping and flow through narrow retentate channels were probably responsible for lowering viability and infectivity. There was no linear relationship between permeate fluxes and cell concentration. There is an optimum pore size both in terms of performance (filtration rate) and cell viability. Physical blockage of large pores by hyphae could explain lower permeate flux rates than those obtained with lower pore sizes membranes.     

Production of a foreign protein product with genetically modified plant cells.

Plant cells (Nicotiana tabacum) were genetically engineered to produce a foreign protein, chloramphenicol acetyltransferase (CAT), and the CAT production from suspension cultures was investigated. Suspension cultures were grown in a shake flask, a stirred fermenter, and a bubble-column fermenter. The CAT production was growth related and the maximum activity was reached during the early stationary phase. A 41-day, semicontinuous stirred fermenter run, consisting of five sequential batch runs, demonstrated long-term CAT production. Continuous CAT production was also accomplished in a bubble-column fermenter at a medium flow rate of 3.1 ml h-1, which was equivalent to a dilution rate of 0.25 day-1.     

Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process

Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA. Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process. Operating conditions including transmembrane pressure, membrane pore size, conductivity of the diafiltration buffer, and plasmid load on the membrane were investigated to maximize RNA clearance. Although direct TFF of clarified lysate removed substantial amounts of RNA, the RNA levels left in the retentate were still significant. Calcium chloride is a potent precipitant of high-molecular-weight RNA. The addition of calcium chloride to the clarified lysate combined with the clearance of low-molecular-weight RNA by TFF resulted in complete RNA removal and high plasmid recovery.     

Continuous cultivation in a chemostat of the phototrophic procaryote,anacystis nidulans,under nitrogen-limiting conditions

Anacystis nidulans was grown photoautotrophically in a chemostat in the presence of light, air and CO2 as the sole carbon source. Either the amount of the nitrogen source in the medium or light intensity were used as growth-limiting parameters. 1. Cells of high glycogen content obtained by pre-incubation under nitrogen starvation conditions maintained their glycogen content during continuous cultivation. Both growth rate and the amount of cell-mass and of glycogen depended on the nitrate content of the medium and the light intensity. The values for the growth rate, the maximal rates of glycogen synthesis and of cell mass formation were 0.1 h-1, 6 mg/l.h and 17 mg/l.h, respectively. 2. Cells without glycogen which had been transferred from an exponentially growing batch culture to chemostat conditions showed increasing rates of growth and of cell mass formation when the light intensity was increased. A determination of specific values resulted in 0.15 h-1 for growth rate and 23 mg/1.h for cell mass formation. 3. The chemostat apparatus is described in detail.     

Vancomycin production is enhanced in chemostat culture with biomass-recycle

Production of the glycopeptide antibiotic vancomycin by Amycolatopsis orientalis ATCC 19795 was examined in phosphate-limited chemostat cultures with biomass-recycle, employing an oscillating membrane separator, at a constant dilution rate (D= 0. 14 h-1). Experiments made under low agitation conditions (600 rpm) showed that the biomass concentration could be increased 3.9-fold with vancomycin production kinetics very similar to that of chemostat culture without biomass-recycle. The specific production rate (qvancomycin) was maximal when the biomass-recycle ratio (R) was 0.13 (D= 0.087 h-1). When the dissolved oxygen tension dropped below 20% (air saturation), the biomass and vancomycin concentrations decreased and an unidentified red metabolite was released into the culture medium. Using increased agitation (850 rpm), used to maintain the dissolved oxygen tension above 20% air saturation, maximum increases in biomass concentration (7.9-fold) and vancomcyin production 1.6-fold (0.6 mg/g dry weight/h) were obtained when R was 0.44 (D= 0.056 h -1) compared to chemostat culture without biomass-recycle. Moreover, at this latter recycle ratio the volumetric vancomycin production rate was 14.7 mg/L/h (a 7-fold increase compared to chemostat culture without biomass-recycle). These observations encourage further research on biomass-recycling as a means of optimising the production of antibiotics.     

A two-stage ultrafiltration and nanofiltration process for recycling dairy wastewater

A two-stage ultrafiltration and nanofiltration (UF/NF) process for the treatment of model dairy wastewater was investigated to recycle nutrients and water from the wastewater. Ultracel PLGC and NF270 membranes were found to be the most suitable for this purpose. In the first stage, protein and lipid were concentrated by the Ultracel PLGC UF membrane and could be used for algae cultivation to produce biodiesel and biofuel, and the permeate from UF was concentrated by the NF270 membrane in the second stage to obtain lactose in retentate and reusable water in permeate, while the NF retentate could be recycled for anaerobic digestion to produce biogas. With this approach, most of dairy wastewater could be recycled to produce reusable water and substrates for bioenergy production. Compared with the single NF process, this two-stage UF/NF process had a higher efficiency and less membrane fouling.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation.

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Product inhibition of immobilized Escherichia coli arising from mass transfer limitation

Mass transfer-limited removal of metabolic products led to product-inhibited growth of Escherichia coli that was immobilized in a model system. Comparison of the growth kinetics of immobilized and free-living cells revealed no further physiological differences between cells in these two modes of existence beyond those manifested in the local concentrations of substrate and product. Bacteria were retained on a microporous membrane in a dense, planar aggregate and were grown anaerobically on a glucose-based minimal medium. Radioisotope labeling of the immobilized cell mass with 35S was used to determine growth kinetic parameters. Growth rates in the immobilized cell layer were measured by an autoradiographic technique which allowed comparison of the size of the growing region with the rate of cell convection caused by growth. Immobilized cell growth rates and growth yields ranged from near maximal (0.56 h-1 and 39 g of dry cell weight/mol of glucose, respectively) to substantially reduced (0.15 h-1 and 15 g/mol). The depression of these kinetic parameters was attributed to product inhibition arising from mass transfer-limited removal of acidic waste products from the cell mass. A simple one-dimensional reaction-diffusion model, which incorporated data on the product-inhibited growth kinetics of free-living cells collected in a product-limited chemostat, satisfactorily predicted product inhibition of immobilized cell growth.     

Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis

Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar -galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular -galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of -galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.     

Dynamic analysis of a microbial process: A systems engineering approach

A general mathematical model of the chemostat system is developed in order to define an experimental program of dynamic testing. A glucose-limited culture ofSaccharomyces cerevisiae was grown in a chemostat using chemically defined medium. The chemostat was perturbed from an initial steady state by changes in input glucose concentration, dilution rate, pH, and temperature. Dynamic responses of cell mass, glucose, cell number, RNA, and protein concentrations were measured. A number of simulation techniques were used in developing a dynamic mathematical model and in comparing the developed model with experimental data as well as the Monod model. The resulting model was found to be quantitatively accurate and superior to the Monod model. The developed model was interpreted in the light of cell physiology. Adjustment of intracellular RNA fraction was found to be rate limiting in acceleration of cell specific growth rate.     

Controlled shear filtration: A novel technique for animal cell separation.

A novel rotary microfiltration technique specifically suited for the separation of animal cells has been developed. The concept allows the independent adjustment of wall shear stress, transmembrane pressure, and residence time, allowing straightforward optimization of the microfiltration process. By using a smooth, conically shaped rotor, it is possible to establish a controlled shear field in which animal cells experience a significant hydrodynamic lift away from the membrane surface. It is shown in preliminary experiments that shear-induced cell-rupture speeds up membrane clogging and that cell debris poses the most significant problem in harvesting of BHK cell cultures by dynamic microfiltration. However, a threshold value of shear stability exists which depends on the frequency of passing the shear field, the residence time in the shear field, as well as on cell status. By operating close to this threshold value, cell viability can be maintained while concentration polarization is efficiently minimized. By applying this concept, it is possible to attain flux rates several times higher compared to conventional crossflow filtration. Controlled shear filtration (CSF) can be used for batch harvesting as well as for cell retention in high cell density systems. In batch harvesting of hIL-2 from rBHK cell culture, a constant flux rate of 290 L h-1 m-2 has been adjusted without indication of membrane clogging or fouling.     

Biological hydrogen production using a membrane bioreactor

A cross-flow membrane was coupled to a chemostat to create an anaerobic membrane bioreactor (MBR) for biological hydrogen production. The reactor was fed glucose (10,000 mg/L) and inoculated with a soil inoculum heat-treated to kill non-spore-forming methanogens. Hydrogen gas was consistently produced at a concentration of 57-60% in the headspace under all conditions. When operated in chemostat mode (no flow through the membrane) at a hydraulic retention time (HRT) of 3.3 h, 90% of the glucose was removed, producing 2200 mg/L of cells and 500 mL/h of biogas. When operated in MBR mode, the solids retention time (SRT) was increased to SRT = 12 h producing a solids concentration in the reactor of 5800 mg/L. This SRT increased the overall glucose utilization (98%), the biogas production rate (640 mL/h), and the conversion efficiency of glucose-to-hydrogen from 22% (no MBR) to 25% (based on a maximum of 4 mol-H(2)/mol-glucose). When the SRT was increased from 5 h to 48 h, glucose utilization (99%) and biomass concentrations (8,800 +/- 600 mg/L) both increased. However, the biogas production decreased (310 +/- 40 mL/h) and the glucose-to-hydrogen conversion efficiency decreased from 37 +/- 4% to 18 +/- 3%. Sustained permeate flows through the membrane were in the range of 57 to 60 L/m(2) h for three different membrane pore sizes (0.3, 0.5, and 0.8 microm). Most (93.7% to 99.3%) of the membrane resistance was due to internal fouling and the reversible cake resistance, and not the membrane itself. Regular backpulsing was essential for maintaining permeate flux through the membrane. Analysis of DNA sequences using ribosomal intergenic spacer analysis indicated bacteria were most closely related to members of Clostridiaceae and Flexibacteraceae, including Clostridium acidisoli CAC237756 (97%), Linmingia china AF481148 (97%), and Cytophaga sp. MDA2507 AF238333 (99%). No PCR amplification of 16s rRNA genes was obtained when archaea-specific primers were used.     

Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 microm ceramic membrane successfully recovered the granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by in-fermenter Benzonase digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and resuspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations.     

Enhancement of chemotaxis in Spirochaeta aurantia grown under conditions of nutrient limitation.

Spirochaeta aurantia M1 cells were grown in a chemostat under conditions of energy and carbon source limitation. The chemotactic responses of the chemostat-grown cells were compared with those of S. aurantia cells grown in batch culture in the presence of excess energy and carbon source. Chemotactic responses were measured by determining the number of cells that entered a capillary tube containing a solution of attractant. S. aurantia cells grown in the chemostat under energy and carbon source limitation exhibited enhanced chemotactic responses and detected lower concentrations of attractant, as compared with cells grown in batch culture. The chemotactic response toward an attractant was specifically enhanced when that attractant was the growth-limiting energy and carbon source. The medium used contained either D-glucose or D-xylose as the sole energy and carbon source. Cells had the greatest chemotactic response toward glucose when grown at a dilution rate (D) of 0.045 h-1 under glucose limitation and toward xylose when grown at D = 0.06 h-1 under xylose limitation. When cells were grown under glucose limitation (D = 0.045 h-1), they sensed concentrations of attractant (glucose) ca. 1,000 times lower than those sensed by batch-grown cells. A similar enhancement of sensing ability (toward xylose) was observed in cells grown under xylose limitation. The results indicated that S. aurantia cells are able to regulate their chemosensory system in response to nutrient limitation. Maximum enhancement of chemotaxis occurs in cells growing at very low concentrations of energy and carbon source. Most likely, this property provides the spirochetes with competitive advantages when the availability of nutrients becomes severely limited in their habitats.