Membrane separations in biotechnology

Membranes have always been an integral part of biotechnology processes. The sterile filtration of fermentation media, purification buffers, and protein product pools is standard practice in industry. Microfiltration is also used extensively for medium exchange and harvest. Ultrafiltration can be found in virtually every biotechnology process. A significant number of mammalian cell processes use filtration as an integral part of the overall strategy for viral clearance. Depth filters have also seen widespread use for the clarification of both mammalian and bacterial feed streams. Improvements in membrane technology are now focused on high-resolution applications, including improved protein-virus separation, protein purification by high-performance tangential flow filtration and enhanced membrane chromatography. These developments will allow membranes to play an important role in the evolution of the next generation of biotechnology processes.     

Use of Membrane Filter Technique in the Microbiological Control for the Brewing Industry

A physical method was developed involving serial filtration with membrane filters for separating yeast cells from bacteria. Such a method eliminates the need for antibiotics previously required to permit differential counting of such populations. All yeast cells filtered were successfully retained and cultivated on a 1.2-mu membrane filter by use of a synthetic medium. All bacteria filtered avoided entrapment on a 1.2-mu membrane filter and were successfully retained and cultivated on a 0.22-mu membrane filter with the same synthetic medium. Final filtrates from these serial filtrations were free from all yeast cells and bacteria when tested with Fluid Thioglycollate Medium.     

Effect of abscisic acid and callus size on regeneration of american and international rice varieties

Embryogenesis and plant regeneration of Texas and international rice, Oryza saliva (L.), varieties (both indica and japonica types) were induced in culture on a regime consisting of the use of ABA or BAP in the subculture medium and small (10 mg) callus pieces on the regeneration medium. Ten 10 mg callus pieces on regeneration medium resulted in a 2- to 10-fold increase in plant regeneration over single 100 mg pieces. Plant regeneration of Texas rice cultivars (Lemont, Rico I, Rexmont and Skybonnet) and Taipei 309 was enhanced by the use of ABA in the subculture medium with a 2-fold and a 3- to 10-fold increase in plant regeneration with 2.6 mgL^–1 and 26 mgL^–1 ABA in the subculture media, respectively. Regeneration of plants from callus of IR36 and IR64 was not enhanced by ABA but by the use of BAP and Trp in the subculture medium or by 2,4-D alone. The subculture medium containing BAP and Trp produced a 5-fold increase in plant regeneration rate from IR64 callus and was equal to subculture medium containing only 2,4-D for IR36 callus. Both Lemont and IR36 were previously reported to be difficult to regenerate or non-regenerative, however, the use of ABA or BAP in the subculture medium, small callus pieces and visual selection of embryogenic callus allowed the regeneration of up to 20 and 22 plants from 100 mg of Lemont and IR36 callus, respectively.Abbreviations ABA abscisic acid - BAP benzylaminopurine - MS Murashige and Skoog - NAA napthaleneacetic acid - Trp tryptophan - 2,4-D 2,4-dichlorophenoxyacetic acid     

Evaluation of Recovery Methods to Detect Coliforms in Water

Various recovery methods used to detect coliforms in water were evaluated by applying the membrane filter chamber technique. The membrane filter chambers, containing pure-culture suspensions of Escherichia coli or natural suspensions of raw sewage, were immersed in the stream environment. Samples were withdrawn from the chamber at regular time intervals and enumerated by several detection methods. In general, multiple-tube fermentation techniques gave better recovery than plating or membrane filtration procedures. The least efficient method of recovery resulted when using membrane filtration procedures, especially as the exposure period of the organisms to the stream environment increased. A 2-h enrichment on a rich, nonselective medium before exposure to selective media improved the recovery of fecal coliforms with membrane filtration techniques. Substantially enhanced recoveries of E. coli from pure-culture suspensions and of fecal coliforms from raw-sewage suspensions were observed when compared with recoveries obtained by direct primary exposure to selective media. Such an enrichment period appears to provide a nontoxic environment for the gradual adjustment and repair of injured cells.     

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.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Use of murine myeloma protein M467 for detecting Salmonella spp. in milk.

This investigation introduces the use of an immunoglobulin A mouse myeloma protein for the detection of Salmonella spp. in milk. The immunoglobulin A protein M467 reacts with flagellin from a wide variety of serotypes. Two assays were developed which used an enzyme-linked immunosorbent assay (ELISA) and M467. Alkaline phosphatase was conjugated to M467 (M467-PH), and the presence of Salmonella dublin was detected by a competitive solid-phase ELISA and a membrane filtration ELISA. The competitive assay competed viable Salmonella spp. found in contaminated milk against polymerized flagellin or whole bacteria fixed to polyvinyl plates for binding by M467-PH. The membrane filtration method utilized a hydrophilic membrane for filtering the bacteria, which were then detected by the reaction with M467-PH and substrate. The sensitivity of the competitive solid-phase ELISA was 10(3) bacteria ml-1, whereas the filter membrane assay required the media containing the bacteria to be cultured in enrichment medium for 4 h before the assay to ensure detection. Either assay could be run within a typical 8-h work day. The filter membrane assay was not suitable for milk due to the high level of natural alkaline phosphatase activity in the liquid food.     

Exposure of Salmonella Enteritidis to chlorine or food preservatives decreases [corrected] susceptibility to antibiotics

Mutants of Salmonella Enteritidis selected following exposure to the sanitizer chlorine or to the preservatives sodium nitrite, sodium benzoate or acetic acid show resistance to multiple antibiotics (tetracycline, chloramphenicol, nalidixic acid, and ciprofloxacin). Complementation experiments with a functional marR restored antibiotic susceptibility of selected mutants to levels similar to wild-type strains, suggesting that mar mutation was responsible for resistance. The multiple antibiotic resistance (mar) operon is a global regulator controlling intrinsic resistance towards structurally and functionally unrelated antibiotics and other noxious agents. Mutants selected after exposure to an inducing agent maintained elevated antibiotic resistance after serial subculture in media void of the inducing agent. Results highlight the importance of monitoring the use of antimicrobial agents to ensure that concentrations capable of inactivating target pathogens are used.     

Recovery Patterns of Spores of Putrefactive Anaerobe 3679 in Various Subculture Media after Heat Treatment

A comparative study was made of the heat resistance of spores of putrefactive anaerobe 3679 grown in two different sporulation media and of the recovery pattern of these spores in several subculturing media after treatment with moist and dry heat. The heat resistance of the spores was characterized in the form of D and z values. The D values were determined by the modified Schmidt method. The z values were established by the graphic method. The results revealed significant differences in D and z values, depending on the type of heat and sporulation and subculture media. Spores grown in beef heart infusion showed higher heat resistance than those grown in Trypticase. Among the seven subculture media used, the largest number of spores was recovered in beef infusion. The magnitude of the D values at 121.1 C obtained with spores heated in moist heat decreased, depending on the subculture medium used, in the following order: beef infusion, pea infusion, yeast extract, liver infusion, Eugonbroth, Trypticase, synthetic medium. With spores subjected to dry heat, D values at 148.9 C decreased with the subculture medium in the following order: beef infusion, yeast extract, pea infusion and liver infusion, Trypticase, Eugonbroth, synthetic medium. The z values obtained with spores subjected to dry heat were approximately double those obtained with moist heat. Their relative magnitude varied slightly, depending on the type of subculture medium used. However, the relative magnitudes of the D values and z values with reference to the subculture media used were different with moist heat from those obtained with dry heat. Two theories are discussed as possible explanations for the logarithmic order of death of bacterial spores. The results obtained in these experiments, together with the findings of other workers, are most compatible with the theory that heat treatment of spores results in an increased rate of random injury to the genetic material of the spores.     

Application of conductance techniques to the detection of thermophilic Campylobacter spp. in river water

A study of basal media identified Campylobacter enrichment broth, with (CEB+) and without (CEB) antibiotic supplement, as a suitable medium for the detection and enumeration of Campylobacter jejuni, C. coli and C. lari within aqueous samples via conductance methodology. Despite apparent differences in conductivity profiles between species in the presence of antibiotics, no significant differences (P<0.05) were detected between detection times for each species tested. CEB+ was successfully employed within a combined enrichment and conductance protocol to the detection of C. jejuni from river water at a concentration of 1 CFU ml⁻¹ from 83% of samples in under 39 h and thus demonstrated an improvement over an applied conventional membrane filtration technique.     

Novel method for selective isolation of actinomycetes

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Novel method for selective isolation of actinomycetes.

A new technique for the selective isolation of actinomycetes from natural mixed microbial populations is described. A nutrient agar medium was overlaid with a 0.22- to 0.45-microns-pore cellulose ester membrane filter, and the surface of the filter was inoculated. During incubation, the branched mycelia of the actinomycetes penetrated the filter pores to the underlying agar medium, whereas growth of nonactinomycete bacteria was restricted to the filter surface. The membrane filter was removed, and the agar medium was reincubated to allow the development of the isolated actinomycete colonies. This procedure selects actinomycetes on the basis of their characteristic mycelial mode of growth, offers a general method for their selective isolation, and does not rely on the use of special nutrient media or of antibacterial antibiotics.     

Influence of physical factors on the growth of insect cells in vitro. II. Sodium and potassium as osmotic pressure regulators of moth cell growth.

The tolerance of a cell line (IMC-HZ-1) from a moth, Heliothis zea, for the monovalent cations Na+ and K+ were defined. Cells shifted to media containing more than 70 mM of K+ showed decreased growth rates. No evidence was obtained for Na+ toxicity. The osmotic pressure tolerances were influenced by the K+ concentration of the medium. The richer the medium was in K+, the narrower was the spectrum of osmotic pressure tolerance. Once the limit of K+ tolerance was exceeded, the rate of decline of growth was linear with respect to further increases in K+. This rate of decline was independent of osmotic pressure. The initial responses of cells during one subculture (2 to 4 population doublings) in media differing from the standard medium (used to maintain the cell line) were not reliable indicators of the growth potential of the cells. Continued subculture in such media resulted in an upward trend in population growth rates in most cases.     

Study of Environmental and Antimicrobial Agents Impact on Features of Bacterial Growth

Continuous, real-time observation of bacterial growth has a great advantage for studying the mechanisms of interactions of various compounds with the bacterial cell membrane. With the use of physical methods, which are specific for assessment of continuous changes in turbidity over time, we have shown that bacterial growth was affected by not only on types of antibiotics and phages, but also by their concentration in media. Low concentration of antibiotics and bacteriophages in media has no effect on the bacterial growth process. Our research has shown that if bacterial cell membrane is not completely saturated with antibiotics membrane sensitive sites (MSS), or bacteriophages free unbounded receptors are remained, bacterial growth continues unimpeded.     

Synergism of thidiazuron and benzyladenine in axillary shoot formation depends on sequence of application in Miscanthus X ogiformis ‘Giganteus’

Shoots of Miscanthus X ogiformis Honda Giganteus transferred from medium with benzyladenine (BA) to thidiazuron (TDZ) formed significantly more axillary shoots than shoots grown continuously on either medium, or transferred from TDZ to BA. Shoot formation on axillary shoots transferred from BA-containing media to media with kinetin or isopentenyladenine (2iP) or transferred from media with TDZ, kinetin or 2iP to media with BA, corresponded to the number of shoots formed in the previous subculture. Shoot formation on shoots transferred from medium containing BA to media containing combinations of BA and/or TDZ increased with increasing concentrations of TDZ in the first subculture, whereas shoot formation in the second and third subculture depended on the BA concentration. When shoots were transferred from media with BA to media with TDZ, the time for shoot formation, as well as shoot size, indicate that the combined effect of BA and TDZ is expressed only during the early phase of the subculture. The results suggest that adenine- and phenylurea-type cytokinins have a common binding site in the plant cell, and a model is proposed.Abbreviations BA 6-benzyladenine - CBP cytokinin-binding protein - 2iP isopentenyladenine - KIN kinetin - MS Murashige & Skoog - TDZ thidiazuron     

Effects of the growth retardant paclobutrazol on large-scale micropropagation of daylily (<Emphasis Type="Italic">Hemerocallis</Emphasis> spp.)

Summary Meristematic clusters were induced from daylily scape explants (pedicel-scape junction) in the presence of the growth retardant Paclobutrazol on semisolid agar medium. Liquid shake culture was used to proliferate meristematic clusters. Highly efficient regeneration of adventitious shoots occurred on clusters after subculture on a 0.8% agar strength semisolid medium with the addition of activated charcoal. Paclobutrazol and sucrose levels in the media were found to significantly affect starch accumulation, growth value, and dry weight percentage of liquid-cultured meristematic clusters. The use of liquid shake cultures for mass proliferation of meristematic clusters followed by regeneration of adventitious shoots on semisolid agar culture could be an efficient system for large-scale micropropagation of daylily.     

Membrane Permeability and the Loss of Germination Factor from Neurospora crassa at Low Water Activities

Neurospora crassa conidia incubating in buffer at low water activities (a(w)) release a germination-essential component as well as 260-nm absorbing and ninhydrin-positive materials, regardless of whether an electrolyte or non-electrolyte is used to reduce a(w). Chloroform and antibiotics known to increase cell-membrane permeability have a similar effect. This suggests that membrane damage occurs in media of low a(w) and that an increase in permeability is responsible for the release of cellular components. The damage caused in media of low a(w) is nonlethal in most cases, and the conidia recover when transferred to nutrient medium.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

Primary Klebsiella identification with MacConkey-inositol-carbenicillin agar.

MacConkey-inositol-carbenicillin agar has successfully been used as a primary selective medium for Klebsiella enumeration. With pure cultures, nearly 100% recovery of Klebsiella was observed by membrane filtration. With environmental samples using membrane filtration, 95% of typical pink- to red-colored colonies were verified as Klebsiella, as opposed to only 1% of yellow background colonies. Recovery of Klebsiella on MacConkey-inositol-carbenicillin agar was as good or better than on mEndo agar LES (Difco Laboratories). Recovery and percent colony confirmation with MacConkey-inositol-carbenicillin agar were greater than for other proposed Klebsiella selective media.     

Recovery of a marker strain of Escherichia coli from ozonated water by membrane filtration.

Selective and nonselective growth media were evaluated at two incubation temperatures, 35 and 44.5 degrees C, for the recovery of a nalidixic acid-resistant marker strain of Escherichia coli ATCC 11775 by membrane filtration from ozonated 0.05 M phosphate buffer (pH 6.9). There were significantly fewer bacteria recovered with the standard m-FC agar when compared with the same growth medium prepared without bile salts and rosolic acid. This effect was particularly noticeable at the elevated incubation temperature of 44.5 degrees C. These findings are contrary to previous work which concluded that the standard American Public Health Association membrane filtration procedure is suitable for recovery of fecal coliform indicator bacteria from ozonated wastewater.