Characteristics of the constituent substrains of Bacillus popilliae growing in batch and continuous cultures.

Continuous culture of Bacillus popilliae was achieved for the first time in a small chemostat. Initially, variable cell yields during steady-state chemostat growth led to a re-examination of growth rates in batch cultures. B. popilliae NRRL B-2309 and a wild strain were both found to be natural mixtures of three substrains characterized by different growth rates and colony morphologies and varying stability. Selected subcultures grown continuously provided data for three different cell production curves. Cell yields were two to three times greater per unit of medium in continuous than in batch culture, and about 1% of slow-growing chemostat cells formed typical spores.     

Analysis of metabolic fluxes in batch and continuous cultures of Bacillus subtilis

It is well recognized that metabolic fluxes are the key variables that must be determined in order to understand metabolic regulation and patterns. However, owing to difficulties in measuring the flux values, evaluation of metabolic fluxes has not been an integral part of the most metabolic studies. Flux values for metabolites of glycolysis, tricarboxylic acid (TCA) cycle, and hexose monophosphate (HMP) pathway were obtained for batch and glucose-limited continuous cultures of Bacillus subtilis by combining the information from the stoichiometry of key biosynthetic reactions with the experimental data on concentrations of glucose and metabolic by-products, CO(2) evolution, and oxygen uptake rates. The results indicate that (1) the metabolic fluxes and energetic yield as well as the extent of flux mismatch in metabolic activity of glycolysis and the TCA cycle reactions can be accurately quantified; (2) the flux through the TCA cycle in continuous culture is much in excess of cell energetic and biosynthetic demands for precursors; (3) for the range of growth rates examined the TCA cycle flux increases almost in proportion to growth rate and is significantly repressed only at very high growth rates of batch cultures; and (4) for continuous cultures the isocitrate dehydrogenase catalyzed reaction of the TCA cycle is the major source of the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) used in biosynthesis. (c) 1993 John Wiley & Sons, Inc.     

Characteristics of the Vegetative Growth of Bacillus popilliae

Growth characteristics of the insect pathogen, Bacillus popilliae Dutky, were studied by propagation in shaken flasks and in 2-liter fermentors. Maximal populations between 5 × 10⁸ and 2 × 10⁹ viable cells per milliliter of culture medium routinely were obtained in incubation periods of 18 to 24 hr at 30 C in a medium composed of 1.5% yeast extract, 0.6% K₂HPO₄, and 0.2% glucose or trehalose. The carbohydrate required for growth in liquid media was fermented with the formation of 2 meq of acid per mmole of carbohydrate utilized; acid products ordinarily were not subsequently metabolized. B. popilliae is an aerobe, and the amount of growth obtained varied with aeration to an optimum at oxygen absorption rates of about 0.5. Maximal populations persist in a culture for periods of only 1 to 4 hr; cessation of growth was followed immediately by rapid death of cultures, so that less than 1% of the cells remained viable after 48 hr, and viability often was lost entirely by the end of 72 hr of incubation. No cytological evidence for spore formation was observed under any growth condition. Death was not associated with lysis of the cells, although extensive granulation ultimately occurred. Continuous neutralizaiton, augmented buffering, various techniques of dialysis, or slow feeding of the carbohydrate did not markedly alleviate the characteristic death of the cultures.     

Effect of carbon dioxide on the formation of α-amylase by Bacillus subtilis growing in continuous and batch cultures

The influence of CO₂ on the formation of α-amylase by Bacillus subtilis NCIB 8646 growing in continuous and batch cultures was investigated. Different levels of CO₂ examined in the batch cultures stimulated the formation of α-amylase, with the highest activity being obtained using 6% CO₂(V /V ). The additions of CO₂ inhibit the growth and division ofvegetative cells of B.subtilis when CO₂ is present in a concentration of more than 3%(V /V ). In chemostat cultures, air containing 8% CO₂ (V /V ) increased the specific enzyme productivity almost three times over the control, without affecting the cell growth. An attempt is made to correlate the obtained results withthe already established theory for the regulation of the α-amylase synthesis.     

Metabolic flux analysis of Escherichia coli expressing the Bacillus subtilis acetolactate synthase in batch and continuous cultures.

Metabolically engineered Escherichia coli expressing the B. subtilis acetolactate synthase has shown to be capable of reducing acetate accumulation. This reduction subsequently led to a significant enhancement in recombinant protein production. The main focus of this study is to systematically examine the effect of ALS in the metabolic patterns of E. coli in batch and continuous culture. The specific acetate production rate of a strain carrying the B. subtilis als gene is 75% lower than that of the control strain (host carrying the control plasmid pACYC184) in batch cultures. The ALS strain is further demonstrated to be capable of maintaining a reduced specific acetate production rate in continuous cultures at dilution rates ranging from 0.1 to 0.4 h-1. In addition, this ALS strain is shown to have a higher ATP yield and lower maintenance coefficient. The metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point reveals that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin.     

Characteristics of N2 fixation in Mo-limited batch and continuous cultures of Azotobacter vinelandii.

Steady-state chemostat cultures of Azotobacter vinelandii were established in a simple defined medium that had been chemically purified to minimize Mo and that contained no utilizable combined N source. Growth was dependent on N2 fixation, the limiting nutrient being the Mo contaminating the system. The Mo content of the organisms was at least 100-fold lower than that of Mo-sufficient cultures, and they lacked the characteristic g = 3.7 e.p.r. feature of the MoFe-protein of nitrogenase. A characteristic of nitrogenase activity in vivo in Mo-limited populations was a disproportionately low activity for acetylene reduction, which was 0.3 to 0.1 of that expected from the rate of N2 reduction. Acetylene was also a poor substrate in comparison with protons as a substrate for nitrogenase, and did not markedly inhibit H2 evolution, in contrast with Mo-sufficient populations. In batch cultures in similar medium or 'spent' chemostat medium inoculated with Mo-limited organisms, the addition of Mo elicited a biphasic increased growth response at concentrations as low as 2.5 nM, provided that sufficient Fe was supplied. In this system V did not substitute for Mo, and Mo-deficient cultures ceased growth at a 25-fold lower population density compared with cultures supplemented with Mo. Nitrogenase component proteins could not be unequivocally detected by visual inspection of fractionated crude extracts of Mo-limited organisms. 35SO42-pulse-labelling studies also showed that the rate of synthesis of the MoFe-protein component of nitrogenase was too low to be quantified. However, the Fe-protein of nitrogenase was apparently synthesized at high rates. The discussion includes an evaluation of the possibility that A. vinelandii possesses an Mo-independent N2-fixation system.     

Extracellular xylanase production by two thermophilic alkali-tolerant Bacillus strains in batch and continuous cultures

Xylanase production of newly isolated thermophilic alkali-tolerant Bacillus sp. strain SP and strain BC was investigated in batch and continuous cultures. Enzyme synthesis was inducible with both strains and was observed only in xylan-containing media. Xylan from oat spelt is a better inducer than xylan from birch for strain Bacillus sp. BC while such difference was not observed for strain SP. Compared with batch cultures xylanase production of both strains increased about two times and its rate became more than four times faster in continuous cultures at a dilution rate of 0.2 h(-1).     

Sporogenicity of yeast autolyzates and casein hydrolyzates for Bacillus popilliae in liquid cultures

Bacillus popilliae NRRL B-2309S forms several million refractile spores/ml in liquid shaken cultures. A suitable medium includes glucose, K₂HPO₄, water, and three selected ingredients-activated carbon, a yeast autolyzate, and a casein hydrolyzate. Only 4 out of 26 lots of commercial yeast autolyzates tried were sporogenic. However, spore formation in the presence of the four was poor and erratic unless a compatible casein hydrolyzate also was present. Five of eight lots of casein hydrolyzates improved the sporogenicity of selected yeast products to various degrees. A comparison of amino acid compositions of yeast and casein lysates sheds no light on differences between suitable and unsuitable ones. Less refined yeast and casein products seem preferable.     

Stability of plasmid-bearing microorganisms in batch and continuous cultures

The stability of five microbial strains bearing a domestic and/or exotic plasmid was investigated in continuous culture to obtain basic information on the fate of genetically engineered microorganisms released in the natural environment.The three strains with an exotic plasmid were constructed by the conjugal or mobilized transfer of conjugative plasmid R100-1 and non-conjugative plasmid RSF2124. Plasmid loss occurred only at the declining growth phase of batch culture of the transconjugants; the ratio of plasmid-free cells was 40–50% at the end of the culture, independent of the strains, whereas the plasmid in the native host cells was maintained at almost 100% of stability.In continuous culture of the transconjugant cells, the population ratio of plasmid-free cells at the pseudo-steady state was between 5–80% depending on the strain. The plasmid-bearing cells were not washed out of the continuous fermentor for 43 generations but maintained their quasi-stable concentration with some degree of oscillation. Simultaneous loss and retransfer of the plasmid from and to its host cells is suggested for the explanation.     

Plasmid stability and alpha-amylase production in batch and continuous cultures of Bacillus subtilis TN106[pAT5

Cell growth and enzyme (alpha-amylase) production characteristics of Bacillus subtilis TN106 containing the recombinant plasmid pAT5 are investigated in batch and continuous cultures using a defined medium with glucose as the limiting nutrient. The batch culture studies demonstrate that the recombinant plasmid, reported earlier(1) to be stably maintained in the host, suffers from segregational and structural instabilities. The structural instability of this strain occurred during culture storage and can be eliminated in bioreactor experiments by using a modified inoculum preparation procedure. Such elimination allows an unbiased investigation of segregational instability via continuous culture studies. Such studies conducted with this fast growing microorganism, in the absence of antibiotic selection pressure, indicate a very efficient glucose utilization (very low residual glucose concentrations) over a wide range of dilution rates (0.16 h(-1) - 0.94 h(-1)). The nearly time-invariant and low residual glucose concentrations at each such dilution rate enable convenient estimation of growth parameters of the host and recombinant cells and frequency of segregational instability from transients in the resulting mixed cultures. The specific alpha-amylase activity exhibits an inverse relationship to the specific growth rate of recombinant cells. The growth of recombinant cells is not affected by the presence of antibiotic (kanamycin). The growth advantage of host cells over recombinant cells diminishes with increasing dilution rate.     

Biodesulfurization of dibenzothiophene by growing cells of Gordonia sp. in batch cultures

A new isolate, identified as Gordonia sp. ZD-7 by 16S rDNA sequence analysis, grew in n-hexadecane containing dibenzothiophene (DBT) which was degraded from 2.8 mM to 0.2 mM within 48 h. Biodesulfurization could be repeatedly performed for more than 190 h, with average desulfurization rates of 5 mmol DBT kg cells (dry wt)⁻¹ h⁻¹.     

Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry

Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse. Using a mixture of two specific fluorescent stains, DiOC6(3) (3,3'-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity. Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC6(3); cells or spores with a depolarised cytoplasmic membrane (B), no staining; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI; and permeablised cells with a disrupted cytoplasmic membrane 'ghost cells' (D), stained with both DiOC6(3) and PI. Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting 'ghost cells' which were associated with the double stained sub-population. It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions. All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch. A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation. Such information could be used to enhance process efficiency.     

Cometabolic degradation of chloroallyl alcohols in batch and continuous cultures

The biodegradation of chloroallyl alcohols by pure and mixed bacterial cultures was investigated. Only 2-chloroallyl alcohol and cis- and trans-3-chloroallyl alcohol served as growth substrate for pure cultures. The other chloroallyl alcohols could be cometabolically degraded during growth on 2-chloroallyl alcohol. Cometabolic degradation of trichloroallyl alcohol, which was the most recalcitrant congener, by a Pseudomonas strain isolated on 2-chloroallyl alcohol resulted in 60% dechlorination. Efficient degradation of a mixture of chloroallyl alcohols in continuous culture could only be achieved in the presence of a satellite population. The mixed culture degraded 99% of the total chloroallyl alcohols added with 71% chloride release. The culture contained strains with a new catabolic potential. The results indicate the importance of mixed cultures and genetic adaptation for efficient chloroallyl alcohol removal.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Characteristics of a New Strain of Bacillus popilliae Sporogenic In Vitro

Conditions are described that led to the isolation of NRRL B-2309M, a strain of Bacillus popilliae which sporulates regularly in laboratory culture. Colonies grown on a medium formulated with yeast extract and the ingredients of Mueller-Hinton with phosphate, trehalose, and agar, produced 20% spores in 10 to 12 days. The quantity and kind of yeast extract determine the extent of sporulation, although there are other requirements for optimal growth and sporulation. Spore inocula free of viable vegetative cells are necessary to maintain sporogenicity since asporogenic substrains arise spontaneously on solid and in liquid media. One such substrain, NRRL B-2309N, is also asporogenic in larvae, but lethal, owing to vigorous vegetative growth. Strain B-2309M is infective when vegetative cells or spores are injected into Japanese beetle larvae but fewer spores are formed in vivo than when infections are caused by NRRL B-2309. The characteristics of four related strains of B. popilliae are tabulated.     

Kinetic studies of the lactic acid fermentation in batch and continuous cultures

The fermentation kinetics of the homofermentative organism Lactobacillus delbrueckii in a glucose-yeast extract medium is studied in both batch and continuous culture under conditions of controlled pH. From a graphical analysis of the batch data, a mathematical model of the process is derived which relates bacterial growth, glucose utilization, and lactic acid formation. The parameters in the model represent the activity of the organism and are a function of pH, having a maximum value at about 5.90. In a continuous stirred tank fermentor (CSTF), the effect of pH, feed concentration, and residence time is observed. The feed medium is a constant ratio of two parts glucose to one part yeast extract plus added mineral salts. An approximate prediction of the steady-state behavior of the CSTF can be made using a method based on the kinetic model derived for the batch case. In making step changes from one steady state to another, the transient response is observed. Using the kinetic model to simulate the transient period, the calculated behavior qualitatively predicts the observed response.     

Invertase biosynthesis by Saccharomyces carlsbergensis in batch and continuous cultures

The biosynthesis of invertase by Saccharomyces carlsbergensis LAM 1068 was studied in relation to its glucose effect at both unsteady and steady states of growth. Experimental correlations between the dilution rate and invertase specific activity (E/X) in chemostat, cultures led to an optimum for the enzyme synthesis at a particular intermediate growth rate. The value of E/X increased from 1.1 (U/mg biomass) in batch cultures to 13 (U/mg biomass) in chemostat cultures. A mutant strain A3 showed the highest value for E/X = 25 (U/mg biomass) at high dilution rates where glucose repression was observed with the wild strain.     

Survival of Lyophilized Bacillus popilliae in Soil

Survival of lyophilized vegetative Bacillus popilliae in dry soil and in soil kept in atmospheres with different relative humidities (RH) was determined. Cells survived for at least 1 year when the RH was 22% or lower, but an RH of 33% or higher reduced viability. At 33% RH, no bacteria were recovered after 5 months of storage; at 42% or above, no bacteria were recovered after 1 month of storage.     

Propagation of Bacillus popilliae in laboratory fermentors

The insect pathogen Bacillus popilliae Dutky causes a fatal milky disease of Japanese beetle larvae. Spores of the bacterium offer a biological means of controlling this insect. While satisfactory sporulation in vitro has not yet been accomplished, conditions have been developed for the proliferation of vegetative cells in shaken flasks and aerated fermentors. Vegetative cultures are maintained by frequent transfer or by lyophilization. Media based on yeast extract are used routinely, but corn steep liquor and casein hydrolyzates afford comparable yields of 5 × 10⁸ cells/ml. in 16–24 hr. Nutritional requirements have been established for growth in a synthetic medium. Oxygen availability affects the pathway of carbohydrate catabolism and is necessary for optimal growth. In rapidly growing cultures, a short period of maximum viability is characteristically followed by rapid death of the cells. When inoculum size and transfer time are suitably manipulated, viable cell yields reach 1–2 × 10⁹/ml. Alternative methods of propagation, including the addition of particulate carbon, and procedures designed either to neutralize acids or to remove metabolic products by dialysis, do not markedly enhance the yield of cells per volume of medium, although viability may be prolonged.