Effect of yeast inoculation rate on the metabolism of contaminating lactobacilli during fermentation of corn mash

Two separate 4 (bacterial concentrations)×6 (yeast concentrations) full factorial experiments were conducted in an attempt to identify a novel approach to minimize the effects caused by bacterial contamination during industrial production of ethanol from corn. Lactobacillus plantarum and Lactobacillus paracasei, commonly occurring bacterial contaminants in ethanol plants, were used in separate fermentation experiments conducted in duplicate using an industrial strain of Saccharomyces cerevisiae, Allyeast Superstart. Bacterial concentrations were 0, 1×10⁶, 1×10⁷ and 1×10⁸ cells/ml mash. Yeast concentrations were 0, 1×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, and 4×10⁷ cells/ml mash. An increased yeast inoculation rate of 3×10⁷ cells/ml resulted in a greater than 80% decrease (P<0.001) and a greater than 55% decrease (P<0.001) in lactic acid production by L. plantarum and L. paracasei, respectively, when mash was infected with 1×10⁸ lactobacilli/ml. No differences (P>0.25) were observed in the final ethanol concentration produced by yeast at any of the inoculation rates studied, in the absence of lactobacilli. However, when the mash was infected with 1×10⁷ or 1×10⁸ lactobacilli/ml, a reduction of 0.7–0.9% v/v (P<0.005) and a reduction of 0.4–0.6% v/v (P<0.005) in the final ethanol produced was observed in mashes inoculated with 1×10⁶ and 1×10⁷ yeast cells/ml, respectively. At higher yeast inoculation rates of 3×10⁷ or 4×10⁷ cells/ml, no differences (P>0.35) were observed in the final ethanol produced even when the mash was infected with 1×10⁸ lactobacilli/ml. The increase in ethanol corresponded to the reduction in lactic acid production by lactobacilli. This suggests that using an inoculation rate of 3×10⁷ yeast cells/ml reduces the growth and metabolism of contaminating lactic bacteria significantly, which results in reduced lactic acid production and a concomitant increase in ethanol production by yeast.     

Superoxide dismutase and the oxygen enhancement of radiation lethality

Escherichia coli B were more susceptible to radiation lethality and showed a greater oxygen enhancement ratio when exposed in dilute suspension (1 × 10⁵ cells/ml) than when exposed in dense suspensions (1 × 10⁹ cells/ml). The oxygen enhancement, seen with dilute suspensions, was diminished by superoxide dismutase, catalase, mannitol, or histidine. Heat-denatured superoxide dismutase was without effect. The results are interpreted as indicating a role for O₂^? plus H₂O₂ in the oxygen enhancement of radiation lethality, and a scheme is proposed which is consistent with the observations.     

Protein production (β-galactosidase) from a baculovirus vector inSpodoptera frugiperda andTrichpolusia ni cells in suspension culture

Protein production capabilities ofTrichpolusia ni (TN 368) cells andSpodoptera frugiperda (Sf9) cells were compared in GTC100 medium in suspension culture using as a vector a genetically engineeredAutographa californica nuclear polyhedrosis virus. TN 368 produces more -galactosidase than Sf9, on a per cell basis (2.2×10⁵ and 1.7×10⁵ units/ 10⁶ cells¹ respectively). In growth experiments serum-free medium supported a higher maximum Sf9 cell density (4±1×10⁶ cells/ml) than the serum- based media (1.5±5×10⁶ cells/ml in GTC100 and 2±1×10⁶ cells/ml in TNM-FH). However, using a cell density of 5×0⁵ cells/ml, the productivity per cell varied, from a low of 4.5×10⁴ units in EX-CELL-400 medium to a high of 7.6×10⁴ units in TNM-FH. The TN 368 cells were twice a large as Sf9 cells and appeared to be more shear sensitive than Sf9 cells.     

Isolation of L-ethionine resistant cell lines in Vigna sinensis L. after mutagen treatment

Chemical mutagenesis was employed for the isolation of variant cell lines resistant to L-Ethionine (L-Eth) in Vigna sinensis L. We have measured cell survival after treatment of Vigna sinensis cell suspensions with different mutagens: Ethyl methanesulfonate (EMS), N-methyl-N-nitrosoguanidine (NTG) and Acridine Orange (AO). NTG was more toxic than EMS and AO.L-Eth resistant colonies were isolated by plating on selective medium after NTG treatment. The frequencies of appearance of resistant cells of MS-3 media supplemented with 20 g/ml L-Eth were 1.3 × 10^-6 to 1.8 × 10^-5. The highest number of L-Eth resistant calli were recorded in cells treated with 10 g/ml NTG. Few resistant colonies also appeared spontaneously from non-mutagenized cultures with a frequency of 2.9 × 10^-7 to 5.7 × 10^-7. However, a number of isolated colonies were discarded after successive retesting. The number of resistant calli dropped from 204 to 22 during successive retests. The significance of these observations has also been discussed.     

Cytotoxicity of trichothecene mycotoxins isolated from Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum in baby hamster kidney (BHK-21) cells

Twenty-six trichothecene mycotoxins produced by Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum were screened for relative cytotoxicity in cultured baby hamster kidney (BHK-21) cells. The relative cytotoxicity was measured as LC₁₀₀. The most cytotoxic trichothecenes were T-2 toxin (5 ng/ml) and the recently isolated 4-propanoyl HT-2 (5 ng/ml) and 3-hydroxy T-2 toxin (5 ng/ml). T-2 tetraol (1 × 10⁴ ng/ml), 8--hydroxytrichothecene (1 × 10⁴ ng/ml), sporotrichiol (2 × 10⁴ ng/ml), 8-oxodiacetoxyscirpenol (6 × 10⁴ ng/ml) and 8-acetyl T-2 tetraol (1 × 10⁵ ng/ml) were the least toxic of the regular trichothecenes. None of the modified trichothecenes or the apotrichothecene were very cytotoxic: 8--hydroxysambucoin (2 × 10³ ng/ml), FS-1 (5 × 10³ ng/ml), 8--hydroxysambucoin (8 × 10⁴ ng/ml) and trichotriol (1 × 10⁵ ng/ml). The modified trichothecenes, FS-2 and FS-3, were not toxic even at 1 × 10⁵ ng/ml. The baby hamster kidney cell bioassay proved to be a very sensitive and reproducible means of screening new trichothecene mycotoxins for relative cytotoxicity.     

Electropermeabilization of dense cell suspensions

This paper investigates the influence of cell density on cell membrane electropermeabilization. The experiments were performed on dense cell suspensions (up to 400 × 10⁶ cells/ml), which represent a simple model for studying electropermeabilization of tissues. Permeabilization was assayed with a fluorescence test using Propidium iodide to obtain the mean number of permeabilized cells (i.e. fluorescence positive) and the mean fluorescence per cell (amount of loaded dye). In our study, as the cell density increased from 10 × 10⁶ to 400 × 10⁶ cells/ml, the fraction of permeabilized cells decreased by approximately 50%. We attributed this to the changes in the local electric field, which led to a decrease in the amplitude of the induced transmembrane voltage. To obtain the same fraction of cell permeabilization in suspensions with 10 × 10⁶ and 400 × 10⁶ cells/ml, the latter suspension had to be permeabilized with higher pulse amplitude, which is in qualitative agreement with numerical computations. The electroloading of the cells also decreased with cell density. The decrease was considerably larger than expected from the differences in the permeabilized cell fractions alone. The additional decrease in fluorescence was mainly due to cell swelling after permeabilization, which reduced extracellular dye availability to the permeabilized membrane and hindered the dye diffusion into the cells. We also observed that resealing of cells appeared to be slower in dense suspensions, which can be attributed to cell swelling resulting from electropermeabilization.     

Foam separation of Escherichia Coli with a cationic surfactant

An experimental investigation of the foam separation of E. coli from distilled water suspension using a cationic surface-active agent, ethylhexadecyldimethyl-ammonium bromide (EHDA-Br) is presented. Results are evaluated in terms of total cell count, using a membrane filtration technique. Cell concentrations in the initial suspensions are varied from 5.0 × 10⁵ to 1.0 × 10⁸ cells/ml. Surfactant concentrations in the initial cell suspensions are varied from 0.015 to 0.040 mg./ml., and foaming times are varied from 2 to 20 min. The residual quantity of cells decreases exponentially with foaming time to about 0.02% of the initial quantity after 20 min. The cell enrichment ratio, varying from 10 to 1,000,000, is an inverse power function of the initial surfactant concentration and an exponential function of foaming time. Foaminess decreases with increasing initial cell concentrations, and for an initial surfactant concentration of 0.030 mg./ml., the residual cell concentration is a linear function of the initial cell concentration.     

Isolation and characterization of pathogens attacking Pomacea canaliculata

Seven microorganisms capable of killing Pomacea canaliculata were isolated from soil samples obtained from various agricultural areas of Thailand. The identification of these microorganisms was performed using microscopic examination and biochemical tests. Five strains were identified as Pseudomonas aeruginosa and were designated P. aeruginosa 19.1, 21.2.1, B1.1, P1 and P2. The other two strains were identified as Pseudomonas fluorescens and were designated P. fluorescens 13.1 and Ct1. Pathogenicity studies of these microorganisms to P. canaliculata (Lamarck) were performed and characterized by LC₅₀ levels. The LC₅₀ levels of non-autoclave-treated and autoclave-treated cell suspensions to P. canaliculata were found to be 3.56 × 10⁴–1.35 × 10⁶ c.f.u./ml and 3.09 × 10⁴ to 1.23 × 10⁶ c.f.u./ml, respectively.     

The rapid loss of viability ofAzotobacter in aqueous solutions

When a low number ofAzotobacter vinelandii 12837 log phase vegetative cells (2 × 10³ cells/ml) were removed from the culture liquid to water of the same temperature, a rapid loss of viability occurred depending on the procedure of washing and suspending. Death was not accompanied by visible lysis and the rate of loss of viability was less at lower temperatures, and in the presence of salts or cell-free filtrates from heavy cell suspensions in water. The die-off was erratic at increased cell concentrations and was accelerated by utilizable energy sources. Cells standing in a favorable ionic solution (0.1% NaCl) do not lose their viability while cells washed by a series of centrifugations with the same ionic solution show a progressive loss of viability with each washing. Phospholipids were found to leach from the cells into the aqueous solutions. Such cell death suggests instability of the cell membrane and the loss of osmotic or ionic control in the cells.This work was supported by grants AI-02830 and GM 600 from the U.S. Public Health Service.     

Culture and Function of Electrofusion-Derived Clonal Insulin-Secreting Cells Immobilized on Solid and Macroporous Microcarrier Beads

In view of the advantages of the bulk production of clonal pancreaticbeta cells, an investigation was made of the growth and insulin secretoryfunctions of an electrofusion-derived cell line (BRIN-BD11) immobilizedon a solid microcarrier, cytodex-1 or a macroporous microcarrier,cultispher-G. For comparison, similar tests were performed usingBRIN-BD11 cells present in single cell suspensions or allowed toform pseudoislets. Similar growth profiles were recorded for eachmicrocarrier with densities of 4.4×10⁵±0.3 cells/ml and4.2×10⁵±0.2 cells/ml achieved using cytodex-1 andcultispher-G, respectively. Cell viability began to decline on day 5 ofculture. Insulin concentration in the culture medium reached a peak of26±2.0 ng/ml and 24±2.2 ng/ml for cells grown oncytodex-1 and cultispher-G, respectively. Cells grown on both types ofmicrocarrier showed a significant 1.5–1.8-fold acuteinsulin-secretory response to 16.7 mmol/l glucose. L-alanine (10 mmol/l) andL-arginine (10 mmol/l) also induced significant 3–4 fold increasesof insulin release. BRIN-BD11 cells immobilized on cytodex-1 or cultispher-Gout-performed single cell suspensions and pseudoislets in terms ofinsulin-secretory responses to glucose and amino acids. A 1.3-fold,2.2-fold and 1.7-fold stimulation of insulin secretion was observed forglucose, L-alanine and L-arginine respectively in single cellsuspensions. Corresponding increases for pseudoislets were1.6–1.8-fold for L-alanine and L-arginine, with no significantresponse to glucose alone. These data indicate the utility ofmicro-carriers for the production of functioning clonal beta cells.     

Immobilization of β-galactosidase retained in yeast: adhesion of the cells on a support

Summary Treatment of Kluyveromyces cells with several polycations partially neutralized their negative charge, even after washing; chitosan at 0.5 mg/ml rendered the zeta potential positive. Adhesion of the cells to various supports (glass, polycarbonate, polystyrene) was promoted by treating the cells or the support with chitosan; this confirms the importance of electrostatic interactions in the adhesion process. When the support was treated, a dense and regular monolayer of adhering cells was obtained; when the cells were treated the adhering layer was more heterogeneous due to floc formation. Cell ghosts of K. lactis were prepared by CHCl₃/ethanol permeabilization and glutaraldehyde treatment. Treating the ghosts with chitosan provided immobilization of 1.9 to 7.3×10⁶ ghosts per cm² according to the support; treating the support allowed to obtain 5.5 to 5.7×10⁶ ghosts per cm². The lactase activity of the immobilized ghosts was found to be comparable to that of ghosts in suspension, i.e. in the range of 0.9 to 2.2 U/mg dry cell weight, at 30°C, corresponding to 0.02 to 0.14 U/cm² of support. A test in microreactor with a lactase solution showed that 50% of the initial activity was still found, after about 25 days of continuous operation at 30°C.     

Denitrification with methane as electron donor in oxygen-limited bioreactors

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10⁷ cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10⁷ cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10⁴ cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. “In vivo”¹³C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification. Received: 19 October 1999 / Received revision: 11 January 2000 / Accepted: 14 January 2000     

High cell density perfusion culture of mouse-human hybridomas

Summary Five mouse-human hybridomas, H2, H3, V1, V2 and V6 cells secreting anti-virus human monoclonal immunoglobulin G (IgG) were cultured in serum-free media at high density in a settling perfusion culture vessel with an inner cell sedimentation zone. The H2, H3 and V6 cells reached a density of 10⁷ cells/ml in 0.5% (w/v) BSA-ITES-eRDF (see Materials and methods). The H2 cells reached only 6.8 × 10⁶ cells/ml in the absence of bovine serum albumin (BSA), but the addition of 0.2% (w/v) Pluronic F68 increased the maximum cell density to 1.1 × 10⁷ cells/ml, which was the same level as in BSA including medium. On the other hand, Pluronic F68 showed no stimulative effect on the growth of H2 cells in static culture. Pluronic F68 also increased the maximum cell density of V2 cells from 4.6 × 10⁶ cells/ml to 6.9 × 10⁶ cells/ml even in the presence of 0.5% (w/v) BSA.     

Cell morphometry and osmolarity as early indicators of the onset of embryogenesis from cell suspension cultures of grain legumes and model systems

Abstract

Whatever the in vitro regeneration pathway, it would be of interest to be able to distinguish regeneration-competent from non-regenerating cells and tissues, and as early in culture as possible, as this would allow a dramatic improvement of biotechnology breeding, particularly for the so-called recalcitrant species. With this aim, we examined a range of genotypes of pea (Pisum sativum) and grass pea (Lathyrus sativus), and of the two model species Medicago truncatula, another legume, and Arabidopsis thaliana. This was done by comparing cell suspension cultures of different ages (young [<6 transfers-old]vs. mature [>6 transfers-old]), densities (dense [>10⁷ cells/ml] or sparse [<10⁶ cells/ml]) and regeneration abilities (non-embryogenic vs. embryogenic), in order to identify early indicators of competence for somatic embryogenesis. All such cell suspensions were subcultured every 14 days and several parameters were assessed every 3–4 days during each 14-day cycle. These included the time course pH and osmolarity of the culture medium, the internal osmolarity of cells, the cell surface and the cell wall thickness (by examining cellulose accumulation in Calcofluor White-stained cells under UV light). As cells underwent embryogenesis they enlarged. Cellulose accumulated in the walls of non-embryogenic cells, but walls became thinner with the onset of embryogenesis, and diminished further as embryos matured. Although medium osmolarity decreased at the onset of embryogenesis, this was never observed for non-embryogenic cell suspensions. Conversely, there was a concomitant increase in intracellular osmolarity for embryogenic cells. Medium pH (analysed with the model species only) was not significantly correlated with regeneration competence of cells. For all genotypes and species, the kinetics of cell wall thickness and cell surface, and that of medium and cell osmolarity were reliable early indicators of the competence of cells to undergo somatic embryogenesis. The implication of these results for biotechnological breeding of grain legumes and for plant regeneration competence in general are discussed.     

Lysis by interleukin 2-stimulated tumor-infiltrating lymphocytes of autologous and allogeneic tumor target cells

Summary Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19⁺ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19⁺ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (n=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 10⁹ normal MNC, obtained by leukapheresis, yielded 5 × 10⁸ A-LAK cells with a total of 5.7 × 10⁵ lytic units of cytotoxicity against K562 and a total of 3.3 × 10⁵ lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.     

Dielectric spectroscopy of mammalian cells

The capacitance of suspensions of CHO and HeLa cells (0.5–3×10⁶ cells/ml) has been measured between 0.2 and 10 MHz. As frequencies decrease, there is a continuous increase in capacitance of both the cell suspension and the spent growth medium free of cells, a phenomenon which is partially attributed to an increased polarisation of the electrodes. At a given frequency, subtraction of the capacitance of the spent medium from that of the cell suspension allows one to determine the capacitance of the cells only. The intensity of this signal varies linearly with the biomass and cell size.At low frequencies such as those used in this study (0.25 MHz), where sensitivity is the highest, concentrations as low as 0.5×10⁶ cells/ml can be accurately measured.Suggestions are made how to make these measures on-line, non-invasive and in real time.     

Testicular Sertoli cells influence the proliferation and immunogenicity of co-cultured endothelial cells

The major problem of the application of endothelial cells (ECs) in transplantation is the lack of proliferation and their immunogenicity. In this study, we co-cultured ECs with Sertoli cells to monitor whether Sertoli cells can influence the proliferation and immunogenicity of co-cultured ECs. Sertoli cells were isolated from adult testicular tissue. ECs were divided into the control group and the experimental group, which included three sub-groups co-cultured with 1 × 10³, 1 × 10⁴ or 1 × 10⁵ cell/ml of Sertoli cells. The growth and proliferation of ECs were observed microscopically, and the expression of vascular endothelial growth factor (VEGF) receptor-2 (KDR) was examined by Western blotting. In another experiment, ECs were divided into the control group, the single culture group and the co-culture group with the optimal concentration of Sertoli cells. After INF-γ and TNF-α were added to the culture medium, MHC II antigen expression was detected by immunofluorescence staining and western blotting; interleukin (IL)-6, IL-8 and soluble intercellular adhesion molecule (sICAM) were measured in the culture medium by ELISA. We demonstrated that 1 × 10⁴ cell/ml Sertoli cells promoted the proliferation of co-cultured ECs more dramatically than that in other groups (P < 0.05). Western blotting showed that 1 × 10⁴ cell/ml of the Sertoli cells was most effective in the up-regulation of KDR expression in the co-cultured ECs (P < 0.05). Sertoli cells can effectively suppress INF-γ-induced MHC II antigen expression in co-cultured ECs compared with single culture group (P < 0.05). TNF-α induced the expression of IL-6, IL-8 and sICAM in ECs. When co-cultured with Sertoli cells, their expressions were significantly lower than in the EC single culture group (P < 0.05). ECs co-cultured with Sertoli cells also did not significantly increase the stimulation index of spleen lymphocytes compared to the single culture group (P < 0.05). Our results suggested that co-culturing with Sertoli cells can significantly promote the proliferation of ECs, accelerate post-transplant angiogenesis, while reduce EC immunogenicity and stimulus to lymphocytes.     

Effects of organic compounds on the degradation ofp-nitrophenol in lake and industrial wastewater by inoculated bacteria

Many microorganisms fail to degrade pollutants when introduced in different natural environments. This is a problem in selecting inocula for bioremediation of polluted sites. Thus, a study was conducted to determine the success of four inoculants to degradep-nitrophenol (PNP) in lake and industrial wastewater and the effects of organic compounds on the degradation of high and low concentrations of PNP in these environments.Corynebacterium strain Z4 when inoculated into the lake and wastewater samples containing 20 µg/ml of PNP degraded 90% of PNP in one day. Addition of 100 µg/ml of glucose as a second substrate did not enhance the degradation of PNP and the bacterium utilized the two substrates simultaneously. Glucose used at the same concentration (100 µg/ml), inhibited degradation of 20 µg of PNP in wastewater byPseudomonas strain MS. However, glucose increased the extent of degradation of PNP byPseudomonas strain GR. Phenol also enhanced the degradation of PNP in wastewater byPseudomonas strain GR, but had no effect on the degradation of PNP byCorynebacterium strain Z4.Addition of 100 µg/ml of glucose as a second substrate into the lake water samples containing low concentration of PNP (26 ng/ml) enhanced the degradation of PNP and the growth ofCorynebacterium strain Z4. In the presence of glucose, it grew from 2×10⁴ to 4×10⁴ cells/ml in 3 days and degraded 70% of PNP as compared to samples without glucose in which the bacterium declined in cell number from 2×10⁴ to 8×10³ cells/ml and degraded only 30% PNP. The results suggest that in inoculation to enhance biodegradation, depending on the inoculant, second organic substrate many play an important role in controlling the rate and extent of biodegradation of organic compounds.Abbreviations PNP p-nitrophenol     

Factors influencing protoplast isolation from Coffea arabica cells

Cultured plant cells such as Coffea arabica L. cells, accumulate low concentration of secondary metabolites. One way to obtain high-producing plant cell cultures is to prepare single cell clones by using protoplast systems. Identification of limiting factors should facilitate the development of an isolation procedure that can generate adequate yields of intact and viable protoplasts Coffea arabica L. suspension cells. The most suitable conditions for protoplasting were as follows: 6 g of fresh tissue were plasmolysed in 100 ml of K ₃ salts (Nagy & Maliga 1976) containing 0.5 M sucrose for 1 h at 24°C. Then, 1 g of preplasmolysed cells were incubated in 10 ml of cellulase R10 (1%), macerozyme R10 (0.8%) and driselase (0.5%) in preplasmolysis medium. The protoplasts were collected and purified after 15 h of lytic reaction in the dark, at 28°C. More than 75% and 95% of the cells were converted into protoplasts when 5 and 8 day-old suspensions respectively were used for the release step. A number of viable protoplasts ranging from 3.5×10⁶ to 4.6×10⁶ P g^-1 fresh weight was obtained corresponding to an increase by a factor 10 to 15 of the protoplast yield obtained by Acuna & De Pena (1991).Abbreviations BAP 6-benzylamino purine - BSA Bovine Serum Albumin - 2,4-d 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - MES 2-(N-morpholino)ethanesulfonic acid - NAA naphthalene acetic acid - PI propidium iodide - PCV Packed Cell Volume - fw fresh weight     

Inductive Effect of Hypoxanthine–Xanthine Oxidase System on Lambda Prophage

Hypoxanthine–xanthine oxidase (HX–XO) system is a classical system that can generate superoxide anions. The inductive effect of the HX–XO system for lambda prophage was investigated in this study. The results showed that the system can induce lambda prophage from the lysogenic state to lytic growth. The inductive effect was directly proportional to the concentration of HX and XO and inversely related to the time of preliminary incubation of HX with XO.The cell density of the lysogenic bacteria also greatly affected the inductive effect. The maximal PFU number of 2.9 × 10⁴ PFU/ml was recorded at 0.86 mM HX, 1.6 × 10^–2 U/ml XO, and a cell density of 10⁸ cells/ml. The inductive effect of the HX–XO system was inhibited in the suspensions by glutathione, superoxide dismutase, and catalase. The results provide evidence that free radicals are the primary factors in the induction of lambda prophage in lysogenic bacteria.