Application of a mathematical model and Differential Evolution algorithm approach to optimization of bacteriocin production by Lactococcus lactis C7

The effect of pH and temperature on cell growth and bacteriocin production in Lactococcus lactis C7 was investigated in order to optimize the production of bacteriocin. The study showed that the bacteriocin production was growth-associated, but declined after reaching the maximum titer. The decrease of bacteriocin was caused by a cell-bound protease. Maximum bacteriocin titer was obtained at pH 5.5 and at 22°C. In order to obtain a global optimized solution for production of bacteriocin, the optimal temperature for bacteriocin production was further studied. Mathematical models were developed for cell growth, substrate consumption, lactic acid production and bacteriocin production. A Differential Evolution algorithm was used both to estimate the model parameters from the experimental data and to compute a temperature profile for maximizing the final bacteriocin titer and bacteriocin productivity. This simulation showed that maximum bacteriocin production was obtained at the optimal temperature profile, starting at 30°C and terminating at 22°C, which was validated by experiment. This temperature profile yielded 20% higher maximum bacteriocin productivity than that obtained at a constant temperature of 22°C, although the total amount of bacteriocin obtained was slightly decreased.     

Effects of physical culture parameters on bacteriocin production by Mexican strains of Bacillus thuringiensis after cellular induction

We have shown previously that in the presence of inducer Bacillus cereus 183, significant increases in bacteriocin production and bactericidal activity of B. thuringiensis occur when the latter is cultivated at pH 7.2, 28°C, and 180 rpm. Here we show that this activity can be further improved when B. thuringiensis is induced with B. cereus 183 and then cultivated with modification of pH, temperature, and agitation. Five native strains of B. thuringiensis, LBIT 269, LBIT 287, LBIT 404, LBIT 420, and LBIT 524 which synthesize, respectively, morricin 269, kurstacin 287, kenyacin 404, entomocin 420, and tolworthcin 524, were cultivated in four different fermentation media. Of these, fermentation in tryptic soy broth (TSB) yielded the highest level of bacteriocin activity (~100–133 FU). Bacteria grown in TSB were induced with B. cereus 183 and cultivated at different pH (6.0, 7.2, 8.0), temperature (26, 28, 30°C), and agitation (150, 180, 210 rpm). Full factorial design was performed and results were analyzed with analysis of variance (ANOVA) and Tukey multiple comparison tests at significant level of α ≤ 0.05 to study the influence of the three variables on bacterial growth and bacteriocin production. Our data show that the highest bacteriocin activity was found with LBIT 269 and LBIT 404 with an increase of ~95–100% compared with induced B. thuringiensis strains cultivated under fixed conditions (pH 7.2, 28°C, 180 rpm), for which the data were set at 0%. The optimal conditions for morricin 269 and kenyacin 404 production were, respectively, pH 8, 30°C, 210 rpm and pH 7.2, 26°C, 210 rpm.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42687

The effects of process conditions and growth kinetics on the production of the bacteriocin sakacin P by Lactobacillus sakei CCUG 42687 have been studied in pH-controlled fermentations. The fermentations could be divided into phases based on the growth kinetics, phase one being a short period of exponential growth, and three subsequent ones being phases of with decreasing specific growth rate. Sakacin P production was maximal at 20 °C. At higher temperatures (25–30 °C) the production ceased at lower cell masses, when less glucose was consumed, resulting in much lower sakacin P concentrations. With similar media and pH, the maximum sakacin P concentration at 20 °C was seven times higher than that at 30 °C. The growth rate increased with increasing concentrations of yeast extract, and the maximum concentration and specific production rate of sakacin P increased concomitantly. Increasing tryptone concentrations also had a positive influence upon sakacin P production, though the effect was significantly lower than that of yeast extract. The maximum sakacin P concentration obtained in this study was 20.5 mg l⁻¹. On the basis of the growth and production kinetics, possible metabolic regulation of bacteriocin synthesis is discussed, e.g. the effects of availability of essential amino acids, other nutrients, and energy. Received: 7 June 1999 / Received revision: 15 September 1999 / Accepted: 17 September 1999     

Temperature management strategy for efficient gene expression in a thermally inducible <Emphasis Type="Italic">Escherichia coli</Emphasis>/bacteriophage system

In a two-phase operation, E. coli containing λSNNU1 (Q ⁻ S ⁻) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.     

Production of hepatitis B core antigen in a stirred tank bioreactor: The influence of temperature and agitation

The influence of temperature and agitation on the growth ofEscherichia coli expressing hepatitis B core antigen (HBcAg) in stirred tank bioreactor were investigated. The highest specific growth rate forE. coli (0.844 h⁻¹) was achieved at a temperature of 37°C and an agitation speed of 250 rpm. The activation energy for the growth of theE. coli strain W3110IQ in the stirred tank bioreactor was estimated to be 11 kcal/mol. The highest protein yield was achieved at a temperature of 44°C and an agitation speed of 250 rpm. The relative protein concentration at 44°C is 30 and 6% higher compared to that at 30 and 37°C, respectively.     

The effects of temperature on the aerobic stability of wheat and corn silages

The aim of this work was to study the effects of temperature on the aerobic stability of wheat and corn silages. Three silage samples from each crop were taken from the faces of six different commercial bunker silos immediately after unloading them. The samples were exposed to air for 3 or 6 days at 10, 20, 30 or 40°C. The most intensive deterioration occurred at 30°C. Samples incubated at 30°C had the highest yeast counts, most prolific CO₂ production and greatest increases in pH. Silage samples exposed to 10 or 40°C remained stable. The duration of exposure had a significant effect on aerobic stability, especially at 30°C. Temperature has a significant effect on silage aerobic stability. In a warm climate, special care should be taken during unloading of silage in order to prevent intensive aerobic deterioration. Journal of Industrial Microbiology & Biotechnology (2002) 28, 261–263 DOI: 10.1038/sj/jim/7000237 Received 12 June 2001/ Accepted in revised form 02 November 2001     

The Influence of <Emphasis Type="Italic">P. fluorescens</Emphasis> Cell Morphology on the Lytic Performance and Production of Phage ϕIBB-PF7A

This study aims at assessing the influence of Pseudomonas fluorescence cell morphology on the effectiveness and production of the lytic bacteriophage ϕIBB-PF7A. P. fluorescens were cultured as rods or as elongated cells by varying the temperature and rotary agitation conditions. Cells presented rod shape when grown at temperatures up to 25°C and also at 30°C under static conditions, and elongated morphology only at 30°C when cultures were grown under agitation. Elongated cells were 0.4 up to 27.9 μm longer than rod cells. Rod-shaped hosts were best infected by phages at 25°C which resulted in an 82% cell density reduction. Phage infection of elongated cells was successful, and the cell density reductions achieved was statistically similar (P > 0.05) to those obtained at the optimum growth temperature of P. fluorescens. Phage burst size varied with the cell growth conditions and was approximately 58 and 153 PFU per infected rod and elongated cells, grown at 160 rpm, at 25°C (the optimal temperature) and 30°C, respectively. Phage adsorption was faster to elongated cells, most likely due to the longer length of the host. The surface composition of rod and elongated cells is similar in terms of outer membrane proteins and lipopolysaccharide profiles. The results of this study suggest that the change of rod cells to an elongated morphology does not prevent cells from being attacked by phages and also does not impair the phage infection.     

Identification and partial characterization of tochicin, a bacteriocin produced by Bacillus thuringiensis subsp tochigiensis

Bacillus thuringiensis subsp tochigiensis HD868 was identified as a bacteriocin producer which exhibited a bactericidal effect against closely related species. This bacteriocin designated as tochicin, was partially purified by 75% ammonium sulfate precipitation followed by subsequent dialysis. This partially purified tochicin showed a narrow antibacterial spectrum of activity against most of 20 typical B. thuringiensis strains and a strain of B. cereus, but not against other bacteria and yeasts tested. The antibacterial activity of tochicin on sensitive indicator cells disappeared completely by proteinase K treatment (1 mg ml⁻¹), which indicates its proteinaceous nature. Tochicin was very stable throughout the range of pH 3.0–9.0 and was relatively heat-stable at 90°C, but bacteriocin activity was not detected after boiling for 30 min. The relationship between cell growth and bacteriocin production was studied in a semi-defined medium. Tochicin activity was detected at the mid-log growth phase, reached the maximum at the early stationary phase, but decreased after the stationary phase. Direct detection of tochicin activity on sodium dodecyl sulfate-polyacrylamide gel suggested it has an apparent molecular mass of about 10.5 kDa. Tochicin exhibited a bactericidal activity against B. thuringiensis subsp thompsoni HD522 in phosphate buffer (pH 7.0). Received 02 December 1996/ Accepted in revised form 25 August 1997     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0–12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain’s genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.

     

Lantibiotic nisin Z fermentative production by Lactococcus lactis IO-1: relationship between production of the lantibiotic and lactate and cell growth

 The influence of several parameters on the fermentative production of nisin Z by Lactococcus lactis IO-1 was studied. Considerable attention has been focused on the relationship between the primary metabolite production of bacteriocin and lactate and cell growth, which has so far not been clarified in detail. Production of nisin Z was optimal at 30°C and in the pH range 5.0–5.5. The addition of Ca²⁺ to the medium showed a stimulating effect on the production of nisin Z. A maximum activity of 3150 IU/ml was obtained during pH-controlled batch fermentation in the medium supplemented with 0.1 M CaCl₂. It was about three times higher than that obtained under the optimal conditions for cell growth and lactic acid production. Received: 12 July 1995/Received revision: 11 September 1995/Accepted: 4 October 1995     

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.     

Conditions for Maximum Enflagellation in Naegleria fowleri1

ABSTRACT. Ameba to flagellate transformation in Naegleria fowleri (Lovell strain) was affected by growth temperature, phase of growth, strain of ameba, culture agitation, enflagellation temperature, enflagellation diluent, and cell concentration. Amebae transformed best when they were grown without agitation and enflagellated with agitation. Regardless of growth temperature (23°, 30°, 37°, and 42°C were tested), amebae transformed best at 37°C. Enflagellation was greatest for cells harvested between 24 h (mid-exponential) and 84 h (late stationary) of growth.     

Effect of temperature on cell growth and production of transparent exopolymer particles by the diatom Coscinodiscus granii isolated from marine mucilage

In the autumn of 2007, marine mucilage caused by the diatom Coscinodiscus granii occurred in the central area of Ariake Sound, Japan, and resulted in damage to fishery. To elucidate the mechanism underlying the outbreak of marine mucilage, we examined the effect of temperature on cell growth and production of transparent exopolymer particles (TEPs) in a culture of this species. Growth and TEP production of C. granii are influenced by temperature. The maximum growth rate (1.63 divisions day⁻¹) and cell yield (1,280 cells mL⁻¹) at all temperatures were obtained at 30°C. Optimal growth rates (>1.15 divisions day⁻¹: ca. 70% of maximum) and cell yield (>900 cells mL⁻¹: ca. 70% of maximum) were observed at temperatures of 25–30°C. TEP production by C. granii depended on whether volume- or cell-related values were considered. The maximum volume-normalized increase rates and concentrations of TEP at all temperatures were observed at 25°C. However, when production rates and concentrations of TEP were normalized to cell numbers, optimal values were measured at 10–15°C. In Ariake Sound, when marine mucilage caused by C. granii occurred, the temperature ranged from 25.0 to 25.4°C. This suggests that growth conditions of C. granii are important factors for production of marine mucilage.     

Evaluation of environmental conditions for production of bacteriocin-like substance by <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain P34

The influence of temperature, pH and media on bacteriocin production by Bacillus sp. P34 was investigated. The effect of temperature and initial pH was evaluated by factorial design and response surface methodology (RSM). Statistical analysis of results showed that, in the range studied, the two variables have a significant effect on bacteriocin production. Response-surface data showed maximum antimicrobial activity production at initial pH values between 6.0 and 8.0 and temperatures between 25 and 37 °C. No relationship between bacterial growth and bacteriocin production was observed. RSM proved to be a powerful tool in optimizing the production of antimicrobial activity by Bacillus sp. P34. When different media were tested, maximum bacteriocin production was observed in soybean protein-based medium, but antimicrobial activity was not achieved by cultivation on fish meal, feather meal, whey and grape waste.     

Submerged Cultivation of a Fungal Mutant Strain Humicola Lutea 120-5 Producing Acid Proteinases

The optimal parameters for the cultivation in 10-l fermenters of a mutant strain Humicola lutea 120-5 were established:temperature 30°C, inoculum size 6%, inoculum age 24 h, aeration rate 0,6 vol/vol · min, medium agitation 620 rpm and cultivation time 72 h. A maximal proteolytic activity of 2000 µg tyrosine liberated from 2%casein ml^?1 culture filtrate min^?1 at pH 3.0 and 40°C was obtained under the fixed conditions. α-Amylase biosynthesis during the cultivation of H. lutea 120-5 was observed but it was insignificant to the 72nd h. It is demonstrated that starch can be used as alternative to glucose carbon source. It is proved that the mutant strain H.lutea 120-5 produced two acid proteinases.     

Flexibility of the metabolism of Corynebacterium glutamicum 2262, a glutamic acid-producing bacterium,in response to temperature upshocks

In order to test the temperature sensitivity of glutamate production metabolism, several temperature shifts, from 33 to 37, 38, 39, 40 or 41°C, were applied to the temperature-sensitive strain, Corynebacterium glutamicum 2262, cultivated in a 24-h fed-batch process. Whereas glucose was entirely dedicated to biomass synthesis when cells were grown at 33°C, applying temperature upshocks, whatever their range, triggered a redistribution of the carbon utilisation between glutamate, biomass and lactate production. Although increasing the culture temperature from 33 to 37, 38, 39 or 40°C resulted in final glutamate titers superior to 80 g/l, temperatures resulting in the best chanelling of the carbon flow towards glutamic acid synthesis were 39 and 40°C. Moreover, this study showed that the higher the temperature, the slower the growth rate and the higher the lactate accumulation. Journal of Industrial Microbiology & Biotechnology (2002) 28, 333–337 DOI: 10.1038/sj/jim/7000251 Received 26 September 2001/ Accepted in revised form 23 February 2002     

Biodegradation of 4-chlorophenol by <Emphasis Type="Italic">Arthrobacter chlorophenolicus A6</Emphasis>: effect of culture conditions and degradation kinetics

Among known microbial species, Arthrobacter chlorophenolicus A6 has shown very good potential to treat phenolic wastewaters. In this study, the levels of various culture conditions, namely initial pH, agitation (rpm), temperature (°C), and inoculum age (h) were optimized to enhance 4-chlorophenol (4-CP) biodegradation and the culture specific growth rate. For optimization, central composite design of experiments followed by response surface methodology (RSM) was applied. Results showed that among the four independent variables, i.e., pH, agitation (rpm), temperature (°C), and inoculum age (h) investigated in this study, interaction effect between agitation and inoculum age as well as that between agitation and temperature were significant on both 4-CP biodegradation efficiency and culture specific growth rate. Also, at the RSM optimized settings of 7.5 pH, 207 rpm, 29.6°C and 39.5 h inoculum age, 100% biodegradation of 4-CP at a high initial concentration of 300 mg l⁻¹ was achieved within a short span of 18.5 h of culture. The enhancement in the 4-CP biodegradation efficiency was found to be 23% higher than that obtained at the unoptimized settings of the culture conditions. Results of batch growth kinetics of A. chlorophenolicus A6 for various 4-CP initial concentrations revealed that the culture followed substrate inhibition kinetics. Biokinetic constants involved in the process were estimated by fitting the experimental data to several models available from the literature.