Mathematical evaluation of plantaricin formation supports an auto-induced production mechanism

The production rate of a bacteriocin, produced by Lactobacillus plantarum TMW1.25 and previously named plantaricin1.25, was studied during pH-constant batch fermentations under various growth media conditions. The growth of L. plantarum and production of bacteriocin during the retardation phase were modelled, using 11 different empirical and mechanistic approaches. The optimal pH for bacteriocin production was 4.5. Among the different nitrogen sources tested, yeast extract was the most important, on the basis of the fact that the maximum growth rate decreased 16% without yeast extract, and only 7.2% or 8.1% without meat extract or peptone respectively. However, the change of nitrogen source did not have a significant effect on bacteriocin production. The progression of plantaricin1.25 production during the retardation phase and growth of L. plantarum TMW1.25 could be described by a structured model in which the bacteriocin concentration induces its own production. Among those models not implementing bacteriocin induction, only the one with an exponential increase of bacteriocin yield per unit biomass was suitable to describe bacteriocin production. Computer-aided evaluation of experimental data appears to be helpful in elucidating the relationship between the growth of lactic acid bacteria and bacteriocin production. Received: 22 May 1998 / Received last revision: 9 November 1998 / Accepted: 14 November 1998     

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     

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     

Studies on the use of a thermotolerant strain of Kluyveromyces marxianus in simultaneous saccharification and ethanol formation from cellulose

 The thermotolerant yeast strain, Kluyveromyces marxianus IMB3, was found to be capable of ethanol production during growth at 45°C on media containing milled paper and exogenously added commercial cellulase. At maximum achievable cellulose concentrations in shake-flask cultures, ethanol production increased to 6.6 g/l at 45°C, representing an overall level of conversion of 21% of the maximum theoretical yield. Subsequent studies involving variations in added cellulase concentrations to the batch systems demonstrated that ethanol yields could be increased to 10 g/l at 45°C, which represented 39% of the maximum theoretical yield. As a result of ethanol production at 45°C in the systems examined, we suggest that the thermotolerant ethanol-producing yeast strain K. marxianus represents a novel candidate for use in simultaneous saccharification and conversion of the resulting substrates to ethanol. Received: 9 June 1994/Received revision: 8 August 1994/Accepted: 12 August 1994     

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     

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     

Effect of culture conditions on growth and esterase production by the moderate thermophile Bacillus circulans MAS2

Growth and esterase production (activity on p-nitrophenyl caprylate) by the newly isolated Bacillus circulans MAS2 bacterial strain were studied. The growth rate at 50°C was high (0.9 h^-1) on LB medium with glucose added. Esterase production followed growth with the majority of activity being intracellular during exponential growth phase. During stationary phase, the esterase activity was released in the culture medium. The strain was able to grow at 35– 55°C with maximum growth rate at 50°C, showing a pattern typical of a moderate thermophile. Growth occurred at pH 6–9 with a maximum at 8, with a similar pattern for the esterase production. Addition of glucose, fructose, sucrose or sodium acetate greatly promoted both growth and esterase production while starch, inulin, tributyrin or glycerol showed no effect. Complex nitrogen sources such as tryptone or yeast extract increased growth and esterase production while mineral sources (ammonium chloride or sulfate), glycine or glutamate showed no effect. An increase of tryptone plus yeast extract and glucose concentrations stimulated growth and esterase production which reached 160 U L⁻¹. Received 17 March 1999/ Accepted in revised form 25 June 1999     

Utilization of protein-hydrolyzed cheese whey for production of β-galactosidase by Kluyveromyces marxianus

We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and 37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS 6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks. Received 20 January 1999/ Accepted in revised form 30 April 1999     

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.     

Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101

Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C. beijerinckii NCIMB 8052. C. beijerinckii BA101 demonstrated a greater increase in solvent production than C. beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium. In 1-l batch fermentations, C. beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose. To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture. Received: 29 September 1998 / Received revision: 13 February 1999 / Accepted: 26 February 1999     

Production, recovery and purification of bacteriocins from lactic acid bacteria

Bacteriocins produced by lactic acid bacteria are a heterogeneous group of peptide inhibitors which include lantibiotics (class I, e.g. nisin), small heat-stable peptides (class II, e.g. pediocin AcH/PA1) and large heat-labile proteins (class III, e.g. helveticin J). Many bacteriocins belonging to the first two groups can be successfully used to inhibit undesirable microorganisms in foods, but only nisin is produced industrially and is licensed for use as a food preservative in a partially purified form. This review focuses on the production and purification of class I and class II bacteriocins from lactic acid bacteria. Bacteriocin production is growth associated but the yield of bacteriocin per unit biomass is affected by several factors, including the producing strain, media (carbohydrate and nitrogen sources, cations, etc.) and fermentation conditions (pH, temperature, agitation, aeration and dilution rate in continuous fermentations). Continuous fermentation processes with cell recycle or immobilized cells can result in a dramatic improvement in productivity over batch fermentations. Several simple recovery processes, based on adsorbing bacteriocin on resins or silica compounds, have been developed and can be used to build integrated production processes. Received: 29 December 1998 / Received revision: 23 April 1999 / Accepted: 23 April 1999     

Towards a reduction in excess sludge production in activated sludge processes: biomass physicochemical treatment and biodegradation

To decrease activated sludge production, microbial cell lysis can be amplified to enhance cryptic growth (biomass growth on lysates). Cell breakage techniques (thermal, alkaline, acid) were studied to generate Alcaligenes eutrophus and sludge lysates and to evaluate their biodegradability. Gentle treatment conditions produced the best results. Complete cell deactivation was obtained for temperatures higher than 55 °C. The release kinetics were similar for temperatures varying from 60 °C to 100 °C. A 20-min incubation was suitable for reaching 80% of the maximum releasable carbon. In thermal-chemical hydrolysis, NaOH was the most efficient for inducing cell lysis. Carbon release was a two-step process. First an immediate release occurred, which was of the same order of magnitude for A. eutrophus and sludge [100–200 mg dissolved organic C (DOC) g total suspended solids (TSS)⁻¹], followed by a post-treatment release. The second step was virtually equivalent to the first for sludge, and weaker for A. eutrophus (<50 mg DOC g TSS⁻¹). The biodegradability of the soluble fraction, both the immediate and the post-treatment carbon release, was investigated. The optimal degradation yield, obtained with sludge cells, reached 55% after 48 h of incubation and 80% after 350 h. The most consistent lysis and biodegradation results occurred at pH 10 and 60 °C after a 20-min incubation. Received: 30 October 1998 / Received revision: 16 February 1999 / Accepted: 20 February 1999     

Enhanced production of penicillin V acylase from Streptomyces lavendulae

At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G. Received: 22 March 1999 / Received revision: 16 June 1999 / Accepted: 20 June 1999     

Characterisation of a novel thermotolerant yeast, Kluyveromyces marxianus var marxianus: development of an ethanol fermentation process

The fermentation characteristics of the novel, thermotolerant, isolate Kluyveromyces marxianus var marxianus were determined to evaluate its aptitude for use in an ethanol production process. Sustainable growth was not observed under anaerobic conditions, even in the presence of unsaturated fatty acid and sterol. A maximum ethanol concentration of 40 g L⁻¹ was produced at 45°C, with an initial specific ethanol production rate of 1.7 g g⁻¹ h⁻¹. This was observed at ethanol concentrations below 8 g L⁻¹ and under oxygen-limited conditions. The low ethanol tolerance and low growth under oxygen-limited conditions required for ethanol production implied that a simple continuous process was not feasible with this yeast strain. Improved productivity was achieved through recycling biomass into the fermenter, indicating that utilising an effective cell retention method such as cell recycle or immobilisation, could lead to the development of a viable industrial process using this novel yeast strain. Received 14 February 1998/ Accepted in revised form 19 May 1998     

Kinetics and characteristics of 70 °C, VFA-grown, UASB granular sludge

We studied in batch reactors the kinetics and characterization of 70 °C, volatile fatty acids (VFAs)-grown, upflow anaerobic sludge blanket granular sludge with 55 and 35 °C sludge as reference. The half-saturation constant (K _s), the inhibition constant (K _i), the maximum specific methane production rate (μ_CH4max), and the inhibition response coefficient (n) of the 70 °C sludge were 6.15 mM, 48.2 mM, 0.132 h⁻¹, and 2.48, respectively, while no inhibition occurred at 55 and 35 °C, where the K _s was 3.67 and 3.82 mM, respectively. At 70 °C, the highest initial specific methanogenic activity (ISMA, 0.311 gCH₄-COD per gram volatile solids per day) on VFAs was about 12–15% lower than that on acetate and three to four times less than the ISMA for the 55 and 35 °C sludge. In the acetate conversion study, residual acetate (79 mg l⁻¹) at 70 °C was three to five times higher than that at 55 and 35 °C. Further, the methane produced as percentage of the acetate consumed at 70 °C (89%) was lower than that at 55 (95%) and 35 °C (97%). At 70 °C, 10% of the ISMA remained after 15 days of starvation as compared to 26% (55 °C) and 92% (35 °C) after 30 days of starvation. Thus, the kinetics of the 70 °C granular sludge seem to differ from those at 55 and 35 °C. Received: 1 February 1999 / Accepted: 20 March 1999     

Conversion of corn fiber to ethanol by recombinant E. coli strain FBR3

Escherichia coli strain FBR3 that is an efficient biocatalyst for converting mixed sugar streams (eg, arabinose, glucose, and xylose) into ethanol. In this report, the strain was tested for conversion of corn fiber hydrolysates into ethanol. Corn fiber hydrolysates with total sugar concentrations of 7.5% (w/v) were prepared by reacting corn fiber with dilute sulfuric acid at 145°C. Initial fermentations of the hydrolysate by strain FBR3 had lag times of approximately 30 h judged by ethanol production. Further experiments indicated that the acetate present in the hydrolysate could not solely account for the long lag. The lag phase was greatly reduced by growing the pre-seed and seed cultures on corn fiber hydrolysate. Ethanol yields for the optimized fermentations were 90% of theoretical. Maximum ethanol concentrations were 2.80% w/v, and the fermentations were completed in approximately 50 h. The optimal pH for the fermentation was 6.5. Below this pH, sugar consumption was incomplete and above it, excess base addition was required throughout the fermentation. Two alternative neutralization methods (overliming and overliming with sulfite addition) have been reported for improving the fermentability of lignocellulosic hydrolysates. These methods further reduced the lag phase of the fermentation, albeit by a minor amount. Received 29 September 1998/ Accepted in revised form 20 February 1999     

Effects of elevated temperature on bacterial community structure and function in bioreactors treating a synthetic wastewater

The impact of elevated temperature on bacterial community structure and function during aerobic biological wastewater treatment was investigated. Continuous cultures, fed a complex growth medium containing gelatin and α-lactose as the principal carbon and energy sources, supported mixed bacterial consortia at temperatures ranging from 25–65°C. These temperature- and substrate-acclimated organisms were then used as inocula for batch growth experiments in which the kinetics of microbial growth and substrate utilization, efficiency of substrate removal, and mechanism of substrate removal were compared as functions of temperature. Bacterial community analysis by denaturing gradient gel electrophoresis (DGGE) revealed that distinct bacterial consortia were supported at each temperature. The efficiency of substrate removal declined at elevated temperatures. Maximum specific growth rates and the growth yield increased with temperature from 25–45°C, but then decreased with further elevations in temperature. Thus, maximum specific substrate utilization rates did not vary significantly over the 40°C temperature range (0.64 ± 0.04 mg COD mg⁻¹ dry cell mass h⁻¹). A comparison of the degradation of the protein and carbohydrate portions of the feed medium revealed a lag in α-lactose uptake at 55°C, whereas both components were utilized simultaneously at 25°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 140–145. Received 09 August 1999/ Accepted in revised form 12 November 1999     

Production of ethanol at 45°C on starch-containing media by mixed cultures of the thermotolerant,ethanol-producing yeast Kluyveromyces marxianus IMB3 and the thermophilic filamentous fungus Talaromyces emersonii CBS 814.70

 The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, was shown to produce ethanol at 45°C on starch-containing media supplemented with a crude amylase preparation derived from the thermophilic, filamentous fungus Talaromyces emersonii CBS 813.70. Ethanol production on media containing 4% (w/v) starch increased to a maximum of 15 g/l with 40 h, and this represented 74% of the maximum theoretical yield. Subsequent experimentation involving growth of both organisms in fermentations on starch-containing media (4% w/v) demonstrated that the mixed-culture system was capable of ethanol production at 45°C with maximum yields at 12 g/l obtained with 65 h. The advantages associated with ethanol production by this system are discussed. Received: 16 May 1994/Accepted: 22 October 1994     

β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997     

A novel method for characterisation of microbial growth kinetics on volatile organic compounds

A novel method for the determination of microbial growth kinetics on hydrophobic volatile organic compounds (VOC) has been developed. A stirred tank reactor was operated as a fed-batch system to which the VOC was continuously fed via the gas phase, assuring a constant VOC concentration in the mineral medium. A flow of air was saturated with the VOC, and then mixed with a further flow of air, to obtain a predetermined VOC concentration. Thus, different VOC concentrations in the mineral medium could be obtained by altering the VOC concentration in the feed gas. The growth kinetics of Xanthobacter autotrophicus GJ10 on 1,2-dichloroethane (DCE) and of Pseudomonas sp. strain JS150 on MonoChloroBenzene (MCB) were assessed using this method. The growth of strain JS150 was strongly inhibited at MCB concentrations higher than 160 mg l⁻¹, and the results were fitted using a piecewise function. The growth kinetics of strain GJ10 were described by the Luong model where maximum growth rate μ_max = 0.12 h⁻¹, substrate saturation constant K _S = 7.8 mg l⁻¹, and maximum substrate concentration S _m (above which growth is completely inhibited) = 1080 mg l⁻¹. Varying nitrogen and oxygen flows enabled the effect of oxygen concentration on the growth kinetics of Pseudomonas JS150 to be determined. Received: 30 November 1998 / Received revision: 19 March 1999 / Accepted: 20 March 1999