Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology

The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol⁻¹. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol⁻¹ at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h. Received: 12 February 1999 / Received revision: 31 May 1999 / Accepted: 4 June 1999     

Generalized model of the effect of pH on lactate fermentation and citrate bioconversion in Lactococcus lactis ssp. Lactis biovar. diacetylactis

An aroma-imparting mesophilic lactic starter (Lactococcus lactis ssp. lactis biovar. diacetylactis) was studied in batch culture in medium with 50 g·l^–1 lactose and 2 g·l^–1 citrate. The effect of pH on the physiology of growth and the production of flavour compounds was investigated with a mathematical model. The specific rates of growth and of lactose fermentation obeyed a law of non-competitive inhibition by lactic acid produced, inhibition increasing as the pH of the medium decreased. The pH thus acted indirectly by increasing the proportion of non-dissociated lactic acid, identified as the inhibiting form of lactic acid. The generalized model, taking into account the effect of pH, was tested using fermentations at pH controlled at different values (4.5–6.5), as well as with a fermentation conducted at non-regulated pH. These simulations supported the working hypotheses. The effect of pH on the fermentation of citric acid resulted in an increase in the maximal specific rate of citrate utilization, in the bioconversion yield, and in the constant of diacetyl and acetoin reduction at acid pH. The production of flavour compounds is a complex phenomenon resulting from the interaction of pH, citric acid concentration, and the physiological state of the cells. These results are discussed with respect to the use of this strain in the preparation of manufactured dairy products.     

Growth and bacteriocin production by Enterococcus faecium DPC1146 in batch and continuous culture

Production of the bacteriocin enterocin 1146 (E1146) by Enterococcus faecium DPC1146 was studied in batch and continuous fermentation. Growth was strongly inhibited by lactic acid. In batch fermentations maximum E1146 activity (2.8 MBU L⁻¹) was obtained in 9 h with 20 g L⁻¹ glucose. Increase in initial glucose concentration did not lead to a proportional increase in E1146 activity. A simple linear model was found to be adequate to explain the relationship between specific bacteriocin production rate and specific growth rate in batch fermentations with initial glucose concentration higher than 20 g L⁻¹. Maximum bacteriocin activity (2.9–3.2 MBU L⁻¹) was obtained in continuous fermentations at dilution rates between 0.12 and 0.17 h⁻¹ and specific bacteriocin production rate increased linearly with dilution rate. Received 31 July 1996/ Accepted in revised form 01 November 1996     

Continuous production of l(+)-lactic acid by Lactobacillus casei in two-stage systems

A two-stage two-stream chemostat system and a two-stage two-stream immobilized upflow packed-bed reactor system were used for the study of lactic acid production by Lactobacillus casei subsp casei. A mixing ratio of D ₁₂/D ₂= 0.5 (D = dilution rate) resulted in optimum production, making it possible to generate continuously a broth with high lactic acid concentration (48 g l⁻¹) and with a lowered overall content of initial yeast extract (5  g l⁻¹), half the concentration supplied in the one-step process. In the two-stage chemostat system, with the first stage at pH 5.5 and 37 °C and a second stage at pH 6.0, a temperature change from 40 °C to 45 °C in the second stage resulted in a 100% substrate consumption at an overall dilution rate of 0.05 h⁻¹. To increase the cell mass in the system, an adhesive strain of L. casei was used to inoculate two packed-bed reactors, which operated with two mixed feedstock streams at the optimal conditions found above. Lactic acid fermentation started after a lag period of cell growth over foam glass particles. No significant amount of free cells, compared with those adhering to the glass foam, was observed during continuous lactic acid production. The extreme values, 57.5 g l⁻¹ for lactic acid concentration and 9.72 g l⁻¹ h⁻¹ for the volumetric productivity, in upflow packed-bed reactors were higher than those obtained for free cells (48 g l⁻¹  and 2.42 g l⁻¹ h⁻¹) respectively and the highest overall l(+)-lactic acid purity (96.8%) was obtained in the two-chemostat system as compared with the immobilized-cell reactors (93%). Received: 4 December 1997 / Received revision: 23 February 1998 / Accepted: 14 March 1998     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Growth-associated synthesis of recombinant human glucagon and human growth hormone in high-cell-density cultures of Escherichia coli

Synthesis of two recombinant proteins (human glucagon and human growth hormone) was investigated in fed-batch cultures at high cell concentrations of recombinant Escherichia coli. The glucose-limited growth was achieved without accumulation of metabolic by-products and hence the cellular environment is presumed invariable during growth and recombinant protein synthesis. Via exponential feeding in the two-phase fed-batch operation, the specific cell growth rate was successfully controlled at the desired rates and the fed-batch mode employed is considered appropriate for examining the correlation between the specific growth rate and the efficiency of recombinant product formation in the recombinant E. coli strains. The two recombinant proteins were expressed as fusion proteins and the concentration in the culture broth was increased to 15 g fusion growth hormone l⁻¹ and 7 g fusion glucagon l⁻¹. The fusion growth hormone was initially expressed as soluble protein but seemed to be gradually aggregated into inclusion bodies as the expression level increased, whereas the synthesized fusion glucagon existed as a cytoplasmic soluble protein during the whole induction period. The stressful conditions of cultivation employed (i.e. high-cell-density cultivation at low growth rate) may induce the increased production of various host-derived chaperones and thereby enhance the folding efficiency of synthesized heterologous proteins. The synthesis of the recombinant fusion proteins was strongly growth-dependent and more efficient at a higher specific growth rate. The mechanism linking specific growth rate with recombinant protein productivity is likely to be related to the change in cellular ribosomal content. Received: 27 May 1997 / Received last revision: 31 October 1997 / Accepted: 21 November 1997     

Modelling the kinetics of growth of Acetobacter aceti in discontinuous culture: influence of the temperature of operation

Acetic acid fermentation is the biochemical process by which, under strict conditions of aerobiosis, Acetobacter aceti oxidises the ethanol contained in alcoholic substrates into acetic acid. This paper studies the effect of temperature on the specific growth rate of the microorganisms (μ _C), in particular, the mathematical modelling of this process, with the aim of developing previous studies of the mathematical relationships between μ _C of A. aceti and the concentrations of substrate (ethanol), product (acetic acid) and dissolved oxygen. Until now this relationship has not been widely studied, and only a few studies have looked at the influence of temperature on growth kinetics of this bacteria. We have developed an extensive experimental system, to determine precisely the influence of temperature on the maximum specific growth rate. Received: 15 July 1997 / Received revision: 7 October 1997 / Accepted: 19 October 1997     

Effects of Ni(II) on respirometric oxygen uptake

The effects of Ni(II), substrate and initial biomass concentrations on biochemical oxygen demand (BOD) were studied by using an electrolytic respirometer. The effects of Ni(II) (2.5, 5.0, 10.0, 25.0 mg/l) and substrate (325, 650, 1300 mg/l as chemical oxygen demand) in a synthetic wastewater with differing initial biomass concentrations (1, 10, 100 mg/l) were investigated. The biomass-to-metal ratio was found to be the most important parameter affecting the measured BOD values. The maximum specific growth rates were calculated and the results of batch respirometric experiments were analysed both by graphical and statistical methods. In statistical analyses, a factorial experimental design approach was followed and results were treated by multiple regression techniques. A mathematical model was developed to express the maximum oxygen uptake in terms of nickel, substrate and initial biomass concentrations and their magnitudes of their effects were compared. The biomass-to-metal ratio was found to be very significant so that another model that expresses oxygen uptake in relation to the biomass-to-metal ratio and also to substrate concentration was developed. Finally, the effect of Ni(II) was demonstrated to depend on both substrate and initial biomass concentrations. This effect was stimulatory at low concentrations of Ni(II), and complete inhibition was never observed even at the highest concentration of Ni(II) studied, which was 25.0 mg/l. Received: 4 January 1997 / Received revision: 10 June 1997 / Accepted: 29 June 1997     

Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium

Specific growth rates (μ) of two strains of Saccharomyces cerevisiae decreased exponentially (R ²>0.9) as the concentrations of acetic acid or lactic acid were increased in minimal media at 30°C. Moreover, the length of the lag phase of each growth curve (h) increased exponentially as increasing concentrations of acetic or lactic acid were added to the media. The minimum inhibitory concentration (MIC) of acetic acid for yeast growth was 0.6% w/v (100 mM) and that of lactic acid was 2.5% w/v (278 mM) for both strains of yeast. However, acetic acid at concentrations as low as 0.05–0.1% w/v and lactic acid at concentrations of 0.2–0.8% w/v begin to stress the yeasts as seen by reduced growth rates and decreased rates of glucose consumption and ethanol production as the concentration of acetic or lactic acid in the media was raised. In the presence of increasing acetic acid, all the glucose in the medium was eventually consumed even though the rates of consumption differed. However, this was not observed in the presence of increasing lactic acid where glucose consumption was extremely protracted even at a concentration of 0.6% w/v (66 mM). A response surface central composite design was used to evaluate the interaction between acetic and lactic acids on the specific growth rate of both yeast strains at 30C. The data were analysed using the General Linear Models (GLM) procedure. From the analysis, the interaction between acetic acid and lactic acid was statistically significant (P≤0.001), i.e., the inhibitory effect of the two acids present together in a medium is highly synergistic. Journal of Industrial Microbiology & Biotechnology (2001) 26, 171–177. Received 06 June 2000/ Accepted in revised form 21 September 2000     

Intra- and extracellular concentrations of glutamate, lactate and acetate during growth of Corynebacterium glutamicum on different media

Corynebacterium glutamicum ATCC 17965 was cultivated in a 4-L batch aerated fermentor with glucose, fructose and mixtures of these two sugars in various proportions as carbon sources and with different concentrations of minerals and vitamins. A multilayer centrifugation technique was devised to obtain cell extracts in order to assess intracellular production of glutamate and partitioning between intracellular and extracellular spaces for lactate and acetate, the main by-products produced during the growth phase. Glutamate production increased with the proportion of glucose in the carbon source. The average value for the intracellular concentration of glutamate obtained with basic glucose medium was increased three-fold when initial concentrations of vitamins and minerals were increased four-fold. In this case, overall production of glutamate (16.3 mM) reached the highest value obtained. Production of acetate was weak on all media types (< 1.6 mm). it was the same for lactate synthesis in media where glucose remained the major carbon source (< 2.3 mm). production of lactate was significantly higher on media where fructose was the main carbon source (> 10 mM to 60 mM). The increase in lactate production and the decrease in glutamate production were correlated to a modification of carbon flux distribution between the metabolic pathways as the fructose proportion was increased. An increase in the concentration of minerals favoured production of glutamate during growth. This was correlated with an increase in the NADPH,H⁺ production rate. Received 16 January 1996/ Accepted in revised form 14 January 1997     

Alginate production by Azotobacter vinelandii DSM576 in batch fermentation

The kinetics of growth and alginate production from glucose in a nitrogen and phosphate-rich medium by Azotobacter vinelandii DSM576 were studied in a laboratory fermenter at pH 7 and 35°C. Batch fermentations were carried out both without control of dissolved oxygen concentration (DO) and at 1, 2, 5 and 10% DO. Although growth was faster at higher DO, maximum biomass concentration was lower. No alginate was produced at 10% DO. Alginate production was faster at 5 and 2% DO but higher alginate concentrations and yields were obtained without DO control. Alginate production was growth-associated at 5% DO, but significant amounts of alginate were produced after growth had stopped at lower DO values. In fermentations without DO control the molecular weight of the polymer reached a maximum (11–17.6 × 10⁴) when specific growth rate was between 0.02 and 0.04 h⁻¹ and residual concentration of ammoniacal nitrogen was between 0.01 and 0.02 g L⁻¹ and then sharply decreased. Received 15 August 1997/ Accepted in revised form 08 January 1998     

Substrate inhibition under stationary growth conditions – nutristat experiments with Ralstonia eutropha JMP 134 during growth on phenol and 2,4-dichlorophenoxyacetate

Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP 134 was continuously grown on phenol and 2,4-dichlorophenoxyacetate at elevated levels of stationary substrate concentration by using the nutristat principle in order to study the physiological impact exerted by these toxic substrates. Growth at stationary concentrations of both the substrates resulted in the reduction of growth efficiency and growth rate. The growth yield data revealed a pronounced dependence on the substrate concentration, and the growth yield increasingly diminished with rising substrate concentration. Inhibition was more pronounced with 2,4-dichlorophenoxyacetate, which reduced the growth yield coefficient by 50% at a substrate concentration of 0.1–0.25 mM. The same effect was obtained with phenol at about 5 mM. The growth rate profile had two distinct phases: after an initially strong reduction, the rate levelled-off at higher substrate concentrations. Standardizing the inhibition profiles, by taking into account the maximum effect after extrapolating the data to zero growth yield, revealed an almost identical pattern with both substrates, indicating some common mechanism. The growth yield data show that an increased amount of energy is required for both growth and maintenance. Homeostatic work was increased by a factor of 8 at 75% inhibition; growth collapsed once this amount of energy was no longer available. The effects are discussed with respect to the properties of these substrates functioning as potential uncouplers of energy conservation. Received: 5 June 1997 / Received revision: 7 July 1997 / Accepted: 12 July 1997     

Influence of pH and lactic acid concentration on Clostridium tyrobutyricum during continuous growth in a pH-auxostat

The aim of this project was to establish the minimal inhibitory concentration (MIC) of lactic acid for growth of Clostridium tyrobutyricum. A pH-auxostat was used to maintain a constant pH and to allow continuous growth at the highest possible rates at fixed, but adjustable concentrations of lactate. By raising the concentration of lactic acid and keeping the pH constant, the growth rate was shown to decrease linearly with increasing lactic acid concentration. The p K _a of lactic acid, measured in the actual growth medium at 37°C, was 3.40 (±0.03). Based on this value, the MIC_undiss values for each pH were estimated. The MIC of total lactic acid (MIC_tot) ranged from 150 mmol l⁻¹ to 1510 mmol l⁻¹ at pH 4.6–6.25, respectively. The corresponding MIC values of undissociated lactic acid (MIC_undiss) ranged from 8.9 to 2.1 mmol l⁻¹ at the same pH values. These results emphasize the importance of a rapid pH decrease and an equally rapid initial lactic acid fermentation of the ensilage, in order to sufficiently suppress clostridial growth.     

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     

Optimal pH control of batch processes for production of curdlan by Agrobacterium species

We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species. The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin. The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters. The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion. By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L⁻¹) compared to that of constant pH operation (36 g L⁻¹). Received 24 November 1998/ Accepted in revised form 17 June 1999     

Phenol inhibition kinetics for growth of Acetobacter aceti on ethanol

Acetobacter aceti have been grown on ethanol under inhibitory conditions created by high concentrations of phenol. A defined medium with no vitamin or amino acid supplements has been used such that ethanol was the sole carbon substrate. The culture temperature was maintained at 30 °C while the pH was manually controlled to fall within the range 4.5–6.0 during ethanol consumption. Growth on ethanol at a few thousand milligrams per litre (below the known inhibitory level) resulted in a maximum specific growth rate of 0.16 h⁻¹ with a 95% yield of acetic acid, followed immediately by acetic acid consumption at a growth rate of 0.037 h⁻¹. Phenol was found to inhibit growth by decreasing both the specific growth rate and the biomass yield during ethanol consumption. On the other hand, the yield of acetic acid during ethanol consumption and the yield of biomass during acetic acid consumption remained constant, independent of phenol inhibition. A model is presented and is shown to represent the phenol-inhibited growth behaviour of A. aceti during both ethanol and acetic acid consumption. Received: 6 November 1998 / Received revision: 8 February 1999 / Accepted: 12 February 1999     

Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration.

The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined. Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45. The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition. In the absence of lactic acid, E. coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid. Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid. In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration. A mathematical model to describe these observations was developed based on a Bĕlehrádek-like model for the effects of water activity and temperature. This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species. Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model. However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.     

Effect of growth rate on α-amylase production by Streptomyces sp. IMD 2679

The α-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown at 40 °C and 55 °C in the presence and absence of cobalt, with an inverse relationship between α-amylase production and growth rate. Highest α-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h⁻¹), at 40 °C in the absence of cobalt, while at the highest growth rate (0.35 h⁻¹), at 55 °C in the presence of cobalt, α-amylase production was decreased to 150 units/ml. As growth rate increased, the rate of specific utilisation of the carbon source maltose also increased, from 46 to 123 μg maltose (mg biomass)⁻¹ h⁻¹. The pattern and levels of α-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression. Received: 17 February 1997 / Received revision: 29 April 1997 / Accepted: 11 May 1997     

Effect of feeding strategy on Zymomonas mobilis CP4 fed-batch fermentations and mathematical modeling of the system

In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass (0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/g glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l) and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would not optimize the process. Received: 30 November 1999 / Received revision: 24 March 2000 / Accepted: 7 April 2000     

Ethanol adaptation in a thermotolerant yeast strain Kluyveromyces marxianus IMB3

The maximum ethanol concentration produced from glucose in defined media at 45°C by the thermotolerant yeast Kluyveromyces marxianus IMB3 was 44 g L⁻¹. Acclimatisation of the strain through continuous culture at ethanol concentrations up to 80 g L⁻¹, shifted the maximum ethanol concentration at which growth was observed from 40 g L⁻¹ to 70 g L⁻¹. Four isolates were selected from the continuous culture, only one of which produced a significant increase in final ethanol concentration (50 ± 0.4 g L⁻¹), however in subsequent fermentations, following storage on nutrient agar plates, the maximum ethanol concentration was comparable with the original isolate. The maximum specific ethanol production rates (approximately 1.5 g (gh)⁻¹) were also comparable with the original strain except for one isolate (0.7 g (gh)⁻¹). The specific ethanol productivity decreased with ethanol concentration; this decrease correlated linearly (rval 0.92) with cell viability. Due to the transience of induced ethanol tolerance in the strain it was concluded that this was not a valid method for improving final ethanol concentrations or production rates. Received 18 July 1997/ Accepted in revised form 19 February 1998