Effect of moderate electric field frequency on growth kinetics and metabolic activity of Lactobacillus acidophilus

Moderate electric fields (MEF) have been previously shown to alter the metabolic activity of microbial cells; thus, the effect of frequency and electric field would be of considerable interest. We investigated herein the effects of MEF frequency on microbial growth kinetics and bacteriocin (Lacidin A) production of Lactobacillus acidophilus OSU 133 during fermentation. The following fermentation treatments were compared: conventional (for 40 h), MEF (1 V cm(-1), for 40 h), combination of MEF (1 V cm(-1), for the first 5 h) and conventional (for 35 h) at various frequency levels (45, 60, and 90 Hz) all at 30 degrees C, and control (conventional) fermentation at 37 degrees C. MEF treatments with purely sinusoidal waveforms at all frequencies at 30 degrees C produced a shorter lag phase than conventional fermentation. However, no lag phase reduction was found for a 60 Hz waveform that contained high-frequency harmonics. There was, however, a significant increase in the bacteriocin production under early MEF treatment at 60 Hz with high-frequency harmonics. On the basis of these observations, the fermentation process is accelerated by applying pure sinusoidal MEF at the early stage of growth while a significant increase in the bacteriocin production occurs when sinusoidal field at 60 Hz with harmonics is applied at the early stage of the growth.     

Effect of moderate electric field frequency and growth stage on the cell membrane permeability of Lactobacillus acidophilus

Changes in growth kinetics and metabolic activity of microorganisms under the presence of a moderate electric field (MEF) have been hypothesized as being due to temporary permeabilization of cell membranes. We investigated herein the effects of frequency and growth stage on cell membrane permeabilization of Lactobacillus acidophilus OSU 133 during MEF fermentation. Cells were stained with two fluorescent nucleic acid stains: the green, nonselective, cell membrane permeable SYTO 9, and the red, cell membrane impermeable propidium iodide (PI). Fluorescence exhibition post‐treatment was assessed using fluorescence microscopy. Total plate counting was done to determine whether or not the permeabilized population represented live cells. Fermentation treatments investigated were conventional (control) and MEF (2 V/cm, 45, 60, 1,000, 10,000 Hz) at 30°C. Studies were conducted at 45 Hz for lag, exponential, and stationary phases of growth. Low frequency MEF treated cells exhibited significantly greater numbers of red cell counts than conventional treatments; further, no significant differences existed in viable counts between MEF and conventional treatments, suggesting that the red counts represent permeabilized live cells. MEF treatments at the early stage of bacterial growth at 45 Hz exhibited the maximum permeabilization followed by treatments at 60 Hz. MEF treated samples at frequencies higher than 60 Hz did not exhibit red fluorescence. Cells at lag phase showed the greatest susceptibility to permeabilization followed by those at exponential phase. No evidence of electroporation was observed during the stationary phase. To our knowledge, these observations provide the first evidence that cell membrane permeabilization occurs under the presence of electric fields as low as those under MEF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi

The influence of growth parameters on the fermentative production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi was studied. The bacteriocin production was greatly affected by carbon and nitrogen sources. Strain A164 produced at least 4-fold greater bacteriocin in M17 broth supplemented with lactose than other carbon sources. The amount of 3% yeast extract was found to be the optimal organic nitrogen source. While the maximum biomass was obtained at 37 degrees C, the optimal temperature for the bacteriocin production was 30 degrees C. The bacteriocin production was also affected by pH of the culture broth. The optimal pH for growth and bacteriocin production was 6.0. Although the cell growth at pH 6.0 was nearly the same level at pH 5.5 and 6.5, the greater bacteriocin activity was observed at pH 6.0. Exponential growth took place only during an initial period of the cultivation, and then linear growth was observed. Linear growth rates increased from 0.160 g(DCW) x l(-1) x h(-1) to 0.245 g(DCW) x l(-1) x h(-1) with increases in lactose concentrations from 0.5 to 3.0%. Maximum biomass was also increased from 1.88 g(DCW) x l(-1) to 4.29 g(DCW) x l(-1). However, increase in lactose concentration did not prolong the active growth phase. After 20 h cultivation, cell growth stopped regardless of lactose concentration. Production of the bacteriocin showed primary metabolic kinetics. However, bacteriocin yield based on cell mass increased greatly during the late growth phase. A maximum activity of 131x10(3) AU x ml(-1) was obtained at early stationary growth phase (20 h) during the batch fermentation in M17L broth (3.0% lactose) at 30 degrees C and pH 6.0.     

Growth kinetics of Lactobacillus acidophilus under ohmic heating

Lactobacillus acidophilus OSU133 was inoculated into MRS broth in a fermenter vessel and incubated at 30, 35, or 45 degrees C with agitation. Incubation temperatures were attained by conventional or ohmic heating. An electrical current at low (15 V) or high (40 V) voltage was used to heat the culture directly during fermentations under ohmic heating. The growth parameters (lag period, minimum generation time, and maximum growth) and changes in pH were determined during fermentation. Metabolic activities (consumption of glucose and production of lactic acid and bacteriocin) were determined during fermentation at 35 degrees C under both heating methods. Lag period for L. acidophilus was affected appreciably by the method of heating, but the magnitude of these changes depended on the fermentation temperature. When fermentation was done at 30 degrees C, lag period decreased by 94% under low-voltage ohmic, compared with conventional, heating methods. Ohmic heating did not change the generation time significantly and caused slight, but significant (p < 0.01) decrease in maximum growth. Therefore, the electric current enhances the early stages, but it inhibits the late stages of growth. Ohmic, compared with conventional, heating resulted in higher final pH and lower bacteriocin activity in the fermented medium. However, ohmic heating at 35 degrees C had minimal effect on glucose utilization and lactic acid production by L. acidophilus. Results show that measurement of the electric current when ohmic heating is done at a constant voltage may be used in monitoring such fermentations. In conclusion, ohmic heating is potentially useful in certain applications related to fermented foods. (c) 1996 John Wiley & Sons, Inc.     

Influence of nutrients on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442

The aim of this study was to investigate the effect of complex nutrients on microbial growth and bacteriocin production, in order to improve bacteriocin synthesis during the growth cycle of Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442. The fermentations were conducted at the optimum pH and temperature for bacteriocin production (pH 5.5+/-0.1 and temperature 25+/-0.1 degrees C). Because of their association with the final biomass, conditions favouring the increase of the produced biomass resulted in the increase of bacteriocin activity in the growth medium. Since the produced final biomass and the final concentration of the bacteriocins were associated with the amount of the carbon (glucose) and nitrogen source, better growth of the lactic acid bacterial strains favoured the increase of the specific bacteriocin production. Additionally, the bacteriocin production was influenced by carbon/nitrogen ratio.     

Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin K

Sakacin K is an antilisterial bacteriocin produced by Lactobacillus sake CTC 494, a strain isolated from Spanish dry fermented sausages. The biokinetics of cell growth and bacteriocin production of L. sake CTC 494 in vitro during laboratory fermentations were investigated by making use of MRS broth. The data obtained from the fermentations was used to set up a predictive model to describe the influence of the physical factors temperature and pH on microbial behavior. The model was validated successfully for all components. However, the specific bacteriocin production rate seemed to have an upper limit. Both cell growth and bacteriocin activity were very much influenced by changes in temperature and pH. The production of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance. Acidity dramatically influenced both the production and the inactivation of sakacin K; the optimal pH for cell growth did not correspond to the pH for maximal sakacin K activity. Furthermore, cells grew well at 35 degrees C but no bacteriocin production could be detected at this temperature. L. sake CTC 494 shows special promise for implementation as a novel bacteriocin-producing sausage starter culture with antilisterial properties, considering the fact that the temperature and acidity conditions that prevail during the fermentation process of dry fermented sausages are optimal for the production of sakacin K.     

Modelling the growth and bacteriocin production by Lactobacillus amylovorus DCE 471 in batch cultivation

A model was set up to describe the production of amylovorin L471 by Lactobacillus amylovorus DCE 471, on a laboratory scale, in which the cells are grown in MRS (deMau-Rogosa-Sharpe) broth. The main features of the dynamic model are : (i) increase of the biomass according to a logistic equation ; (ii) non-growth-associated consumption of substrate (maintenance metabolism) ; and (iii) primary metabolite kinetics for the bacteriocin production. The main purpose was to set up a simple empirical model to examine growth and bacteriocin production in different conditions. Parameters estimated from a fermentation with 20 g l⁻¹ glucose (w/v) could be used to predict the evolution of cell dry mass, glucose and lactic acid concentration of fermentations, performed with 5, 30, 40 and 60 g l⁻¹ initial glucose. The influence of the operating temperature (30, 37 and 45 °C) on the model parameters was also investigated. The proposed model was able to describe growth and bacteriocin production in all cases. The specific bacteriocin production rate was found to vary strongly with temperature, with 30 °C as the best value. Variation of the operating temperature from 37 to 30 °C appeared to significantly enhance the specific bacteriocin production.     

Distribution of soil carbon and microbial biomass in arable soils under different tillage regimes

We have measured total soil organic carbon (SOC), dissolved organic carbon (DOC), and microbial lipid contents (as indices of microbial biomass and community structure), and their distributions to 60 cm depth in soils from replicated medium-term (2003?C2008) experimental arable plots subject to different tillage regimes in Scotland. The treatments were zero tillage (ZT), minimum tillage (MT; cultivation to 7 cm), the conventional tillage (CT) practice of ploughing to 20 cm, and deep ploughing (DP) to 40 cm depth. In the 0?C30 cm depth range, SOC content (corrected for bulk density differences between tillage treatments) was greatest under ZT and MT, but over 0?C60 cm depth the SOC contents of these treatments were similar to the CT and DP treatments. DOC concentrations declined with increasing depth in ZT and MT above 20 cm, but there were no significant differences with depth in the CT and DP treatments. Beneath 20 cm, there was little change in DOC concentration with depth for all treatments, although for the MT treatment, there was less DOC beneath the depth of cultivation. The total microbial biomass decreased with increasing depth over the 0?C60 cm range in the ZT and MT treatments, whereas it decreased with depth only below 30?C40 cm in the CT and DP treatments. The microbial biomass was significantly different only between 0?C5 cm in the ZT, CT and DP treatments, but not for other depths between all treatments. The bacterial biomass was greater in the ZT treatment than in MT, CT and DP near the soil surface, but not significantly different over the whole profile (0?C60 cm). The fungal biomass decreased with depth in the ZT and MT treatments over the whole 0?C60 cm depth range, whereas it decreased with depth only below 20 cm in the CT and DP treatments.     

Producton of single-cell protein from cellulose by Aspergillus terreus

Cellulose fermentation studies were conducted with a thermotolerant strain of Aspergillus terreus. Batch cultivation of A. terreus using purified or complex cellulose showed that 80-88% of the available cellulose was utilized in 30-36 h with an average doubling time of 7.5-8.3 h. The protein content in the biomass ranged from 23 to 38%. Semicontinuous cultivation studies, in which 90% of the biomass was withdrawn at the end of growth cycle, indicated that 84% of added cellulose was utilized with the biomass containing 32% crude protein. No loss in cellulose consumption, growth rate, or protein production occurred through two growth cycles. Continuous cultivation of A. terreus showed that 78-84% cellulose consumption occurred over growth temperatures ranging from 35 to 45 degrees C. Maximum specific growth rates (0.14 h(-1)) occurred at 40 and 45 degrees C with a minimum doubling time of 4.9 h.     

保护性耕作对土壤微生物量及活性的影响

研究保护性耕作对土壤微生物特性的影响对于土壤管理具有重要意义。试验研究了保护性耕作对麦田土壤微生物量碳、活跃微生物量、土壤呼吸、呼吸商的影响。前3项采用的方法分别是:基质诱导呼吸法、呼吸曲线数学分析法和CO2释放量法。结果表明,保护性耕作土壤微生物量碳0～10cm土层大于10～20cm土层,而常规耕作两土层间无明显差异。秸秆还田在播种前、越冬期和起身期能显著提高土壤微生物量碳,而开花期和收获期则降低土壤微生物量碳。少耕还田10～20cm土层微生物具有较强的养分调控作用。保护性耕作利于0～10cm土层活跃微生物量的提高。秸秆还田和保护性耕作在耕作作业初期(越冬期和起身期)能增强土壤呼吸速率;在耕作作业后期(开花期和收获期)能显著降低土壤呼吸速率。免耕秸秆覆盖在10～20cm土层呼吸商较高,而常规耕作无秸秆还田在0～10cm土层呼吸商较高。土壤微生物量碳和呼吸商是衡量土壤微生物特性的重要指标。     

Carnocin KZ213 produced by<Emphasis Type="Italic"> Carnobacterium piscicola</Emphasis> 213 is adsorbed onto cells during growth. Its biosynthesis is regulated by temperature,pH and medium composition

Carnocin KZ213 is an antilisterial bacteriocin produced by Carnobacterium piscicola 213. The effects of pH and temperature were studied during batch fermentation in MRS* medium (modified MRS without ammonium citrate or sodium acetate). The optimal pH for growth is between 6 and 7. The maximum bacteriocin productivity in the supernatant occurs at pH 7. Operating at controlled pH increases the volumetric activity of the free bacteriocin by 8- to 16-fold, compared with uncontrolled pH. No bacteriocin production is observed below pH 6.5. Temperature has a dramatic effect on carnocin KZ213 production. Growth is optimal at 25 °C and 30 °C, although no bacteriocin production is detected at 30 °C. Also, bacteriocin production is observed at 25 °C in MRS*, but not in complex APT broth, where growth is optimal. The presence of glucose as a carbon and/or energy source is important for carnocin KZ213 synthesis. Hence, bacteriocin synthesis is regulated by temperature, carbon source and medium composition. Quantification studies of bacteriocin adsorbed onto producer cells show that the majority of the carnocin KZ213 secreted is adsorbed onto the producer cells during growth. Only 15% of the total bacteriocin produced is detected in the cell-free supernatant at the end of growth.     

Production and pH-Dependent Bactericidal Activity of Lactocin S, a Lantibiotic from Lactobacillus sake L45

The amount of lactocin S activity in a growing culture depends on the growth stage of the bacteria, the pH of the medium, the presence of ethanol, and the aeration of the culture. We observed the highest levels of bacteriocin activity in the early stationary growth phase of cultures at 30 deg C. When Lactobacillus sake L45 was grown in a fermentor at pH 5, it produced 2,000 to 3,000 bacteriocin units per ml, which represented an 8- to 10-fold increase in bacteriocin production compared with production during batch culture fermentation. Less than 10% of this level of bacteriocin activity was observed during fermentation at pH 6.0. When 1% ethanol was included in the growth medium, a two- to fourfold increase in the bacteriocin yield was observed. Aerating the culture during growth almost completely eliminated the production of active bacteriocin. Our results also showed that lactocin S-mediated killing of target cells depended on the pH of the culture. The pH had to be less than 6 in order to obtain a bactericidal effect with lactocin S-sensitive cells. At pH values greater than 6, lactocin S had no apparent effect on sensitive cells.     

Influence of pH on the production of enterocin 1146 during batch fermentation

The production of enterocin 1146, a bacteriocin from Enterococcus faecium DPC1146, was studied during batch fermentation at pH 5, 5.5, 6 and 6.5. The bacteriocin was produced throughout the growth of the micro-organism, showing primary metabolite kinetics. Bacteriocin production stopped at the end of growth and was followed by a decrease in activity due primarily to adsorption on the cells of the producer. The optimal pH for enterocin 1146 production was 5.5, because of higher bacteriocin yield per unit of biomass and slower adsorption/degradation, while optimal pH for growth was between 6.0 and 6.5.     

Evaluation of Various Unstructured Kinetic Models for the Production of Protease by Bacillus sphaericus MTTC511

Bacillus sphaericus MTCC511 was used for the production of protease in submerged batch fermentation. Maximum protease activity of 1010 U/L was obtained during a fermentation period of 24 h under optimized conditions of 30 °C in a medium with an initial pH of 7 and at a shaking rate of 120 rpm. The maximum biomass obtained in the batch fermentation was 2.55 g/L after 16 h. Various unstructured models were analyzed to simulate the experimental values of microbial growth, protease activity and substrate concentration. The unstructured models, i.e. the Monod model for microbial growth, the Monod incorporated Luedeking‐Piret model for the production of protease and the Monod‐incorporated modified Luedeking‐Piret model for the utilization of substrate were capable of predicting the fermentation profile with high coefficient of determination (R²) values of 0.9967, 0.9402 and 0.9729, respectively. The results indicated that the unstructured models were able to describe the fermentation kinetics more effectively.     

温度对谷胱甘肽分批发酵的影响及动力学模型

研究了24～32℃范围内产朊假丝酵母生产谷胱甘肽的分批发酵过程,发现较高温度对细胞生长有促进作用,而较低温度则更有利于谷胱甘肽产量的提高。应用改进的Logistic和LuedekingPiret方程分别对细胞生长动力学和谷胱甘肽合成动力学进行了模拟,得到不同温度下各种动力学参数。在此基础上,进一步研究了温度同细胞生长动力学参数之间的内在联系,得到谷胱甘肽分批发酵过程中细胞浓度的变化同温度以及底物浓度之间的一般关系式：dX-dt＝［0.0224(T+1.7)］2X(1-X/X_max)1+S｛8.26×10.6×exp［-31477/R/(T+273)］｝。验证实验结果表明,该模型具有很好的适用性。     

Effect of temperature on Brettanomyces bruxellensis: metabolic and kinetic aspects

The effect of temperatures ranging from 15 to 35 degrees C on a culture of Brettanomyces bruxellensis was investigated in regards to thermodynamics, metabolism, and kinetics. In this temperature range, we observed an increase in growth and production rates. The growth behavior was well represented using the Arrhenius model, and an apparent activation energy of 16.61 kcal/mol was estimated. A stuck fermentation was observed at 35 degrees C as represented by high cell death. The carbon balance established that temperature had no effect on repartition of the glucose consumption between biomass and products. Hence, the same biomass concentration was obtained for all temperatures, except at 35 degrees C. Moreover, using logistic and Luedeking-Piret models, we demonstrated that production rates of ethanol and acetic acid were partially growth associated. Parameters associated with growth (alpha eth and alpha aa) remained constant with changing temperature, whereas, parameters associated with the population (beta eth and beta aa) varied. Optimal values were obtained at 32 degrees C for ethanol and at 25 degrees C for acetic acid.     

Fermentation efficiency of thermally dried kefir

Three thermal drying methods (conventional, vacuum and convective) were used for drying of kefir biomass and their effect on cell viability, fermentation rate and other kinetic parameters of lactose and whey fermentation were studied. Convective drying rate was higher than conventional and even higher than vacuum at each studied temperature (28, 33 and 38 degrees C). After that, fermentations were performed by kefir biomass dried by the three drying methods. Ethanol concentration, ethanol productivity and ethanol yield are higher in whey fermentations performed by kefir biomass dried with convective drying method. Regarding lactic acid production, fermentation performed by kefir biomass dried with conventional drying method gave higher concentrations, compared to other drying methods. Storage of kefir biomass convectively dried at 33 degrees C for 4months, without any precaution decreases its fermentability and thus reduces ethanol (31%) and lactic acid productivity (20%), but remains a promising technology, since a significant part of its initial fermentative activity is retained.     

Optimization of enterocin P production by batch fermentation of Enterococcus faecium P13 at constant pH

The influence of pH on growth, enterocin P production and glucose consumption by Enterococcus faecium P13 was studied during anaerobic batch fermentation in MRS broth at 32 degrees C in a fermentor. Growth and glucose consumption were maximal at pH 7.0. Enterocin P production displayed primary metabolite kinetics and was strongly dependent on pH. A maximum antimicrobial activity of 1,949 bacteriocin units (BU) ml(-1) was obtained at pH 6.0, which represented a four-fold increase compared with the antimicrobial activity obtained without pH regulation. The pH exerted a marked effect on the decrease in bacteriocin activity, with the decrease being maximal at pH 7.0. In this report, we propose models for the growth of E. faecium P13 as well as enterocin P production and inactivation. Enterocin P production decreased when potentially stress-inducing compounds (NaCl or ethanol) were included in the growth medium.     

The Curing Agent Sodium Nitrite, Used in the Production of Fermented Sausages, Is Less Inhibiting to the Bacteriocin-Producing Meat Starter Culture Lactobacillus curvatus LTH 1174 under Anaerobic Conditions

Curvacin A is a listericidal bacteriocin produced by Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.     

渤海泥质海岸典型防护林土壤微生物量季节动态变化

土壤微生物生物量碳、氮是研究土壤肥力、土壤养分转化、循环以及环境变化的重要指标。研究渤海泥质海岸白榆、刺槐、白蜡、群众杨、辽宁杨纯林和辽宁杨刺槐混交林及当地自然生灌草地土壤微生物生物量碳、氮的季节动态及与土壤养分含量变化的关系,以期为沿海防护林树种的选择及林地管理提供科学依据。结果表明:造林能显著增加土壤微生物生物量含量,其中白榆(25 a)土壤微生物生物量碳、氮最高,是对照的2.50倍和2.09倍。0—10 cm土壤层微生物生物量碳、氮大于10—30 cm土层,季节动态变化差异显著。在0—10 cm土层内,渤海泥质海岸典型防护林土壤微生物生物量碳、氮季节动态多表现为春秋两季较高,夏季较低的"V"字型变化;在10—30 cm土层内,防护林土壤微生物生物量碳季节变化规律与0—10 cm土层一致,表现为夏季较低春秋较高的"V"字型,微生物生物量氮主要表现有"V"字型、倒"V"字型与直线型3种变化形式。在0—30 cm土层内,白榆(25 a)、刺槐、白蜡、群众杨、辽宁杨刺槐混交林、白榆(10 a)、辽宁杨及灌草地微生物生物量碳对土壤有机碳的平均贡献率分别为1.59%、1.68%、1.42%、1.54%、2.29%、1.80%、2.02%和1.12%,土壤微生物生物量氮对土壤全氮的平均贡献率分别为1.85%、1.30%、1.08%、1.35%、2.49%、1.57%、2.08%和2.32%。不同类型防护林地土壤微生物量碳、氮之间显著正相关,它们与土壤全氮、有机碳显著正相关,与土壤电导率显著负相关,另外,土壤微生物量碳还与土壤速效磷含量显著正相关。从不同土层微生物量碳、氮季节动态来看,造林可以增加泥质海岸土壤微生物生物量,但是夏季地下水位升高,盐碱上扬,加之树木生长大量利用养分,土壤微生物生物量夏季较低。综合分析土壤微生物生物量和土壤营养库的贡献率,白榆纯林和辽宁杨刺槐混交林更有利于泥质海岸土壤微生物群落功能恢复和营养固定。