Factors affecting the antibacterial activity of the ruminal bacterium,Streptococcus bovis HC5

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h(-1)) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2-1.2 h(-1)) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h(-1)) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h(-1)) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml(-1)) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application.     

The diversion of lactose carbon through the tagatose pathway reduces the intracellular fructose 1,6-bisphosphate and growth rate of Streptococcus bovis

Twenty strains of Streptococcus bovis grew more slowly on lactose (1.21 ± 0.12 h⁻¹) than on glucose (1.67 ± 0.12 h⁻¹), and repeated transfers or prolonged growth in continuous culture (more than 200 generations each) did not enhance the growth rate on lactose. Lactose transport activity was poorly correlated with growth rate, and slow growth could not be explained by the ATP production rate (catabolic rate). Batch cultures growing on lactose always had less␣intracellular fructose 1,6-bisphosphate (Fru1,6P ₂) than cells growing on glucose (6.6 mM compared to 16.7 mM), and this difference could be explained by the pathway of carbon metabolism. Glucose and the glucose moiety of lactose were metabolized by the Embden-Meyerhoff-Parnas (EMP) pathway, but the galactose moiety of lactose was catabolized by the tagatose pathway, a scheme that by-passed Fru1,6P ₂. A mutant capable of co-metabolizing lactose and glucose grew more rapidly when glucose was added, even though the total rate of hexose fermentation did not change. Wild-type S. bovis grew rapidly with galactose and melibiose, but these galactose-containing sugars were activated by galactokinase and catabolized via EMP. On the basis of these results, rapid glycolytic flux through the EMP pathway is needed for the rapid growth (more than 1.2 h⁻¹) of S.␣bovis. Received: 3 June 1997 / Received revision: 10 September 1997 / Accepted: 6 January 1998     

The ability of non-bacteriocin producing Streptococcus bovis strains to bind and transfer bovicin HC5 to other sensitive bacteria

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5), but S. bovis JB1 does not have antimicrobial activity. Preliminary experiments revealed an anomaly. When S. bovis JB1 cells were washed in stationary phase S. bovis HC5 cell-free culture supernatant, the S. bovis JB1 cells were subsequently able to inhibit hyper-ammonia producing ruminal bacteria (Clostridium sticklandii, Clostridium aminophilum and Peptostreptococcus anaerobius). Other non-bacteriocin producing S. bovis strains also had the ability to bind and transfer semi-purified bovicin HC5. Bovicin HC5 that was bound to S. bovis JB1 was much more resistant to Pronase E than cell-free bovicin HC5, but it could be inactivated if the incubation period was 24 h. Acidic NaCl treatment (100 mM, pH 2.0) liberates half of the bovicin HC5 from S. bovis HC5, but it did not prevent bovicin HC5 from binding to S. bovis JB1. Acidic NaCl liberated some bovicin HC5 from S. bovis JB1, but the decrease in activity was only 2-fold. Bovicin HC5 is a positively charged peptide, and the ability of S. bovis JB1 to bind bovicin HC5 could be inhibited by either calcium or magnesium (100 mM). Acidic NaCl-treated S. bovis JB1 cells were unable to accumulate potassium, but they were still able to bind bovicin HC5 and prevent potassium accumulation by untreated S. bovis JB1 cells. Based on these results, bovicin HC5 bound to S. bovis JB1 cells still acts as a pore-forming lantibiotic.     

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)⁻¹(min)⁻¹. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K _s ) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a K_s of 0.20 mM, but the K_s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport. Received: 22 January 1996 / Accepted: 18 March 1996     

The Production of Bacillus cereus Enterotoxins Is Influenced by Carbohydrate and Growth Rate

Enterotoxin production is a key factor in Bacillus cereus food poisoning. Herein, the effect of the growth rate (μ) on B. cereus toxin production when grown on sucrose was studied and the Hemolytic BL enterotoxin (HBL) and nonhemolytic enterotoxin (Nhe) production by B. cereus was compared according to carbohydrate at μ = 0.2 h⁻¹. The anaerobic growth was carried out on continuous cultures in synthetic medium supplemented with glucose, fructose, sucrose, or an equimolar mixture of glucose and fructose. Concerning the HBL and Nhe enterotoxin production: (1) the highest enterotoxin production has occurred at μ = 0.2 h⁻¹ when growing on sucrose; (2) HBL production was repressed when glucose was consumed and the presence of fructose (alone or in mixture) cancelled glucose catabolite repression; (3) the consumption of sucrose increased Nhe production, which was not affected by the catabolite repression. Furthermore, analysis of the fermentative metabolism showed that whatever the μ or the carbon source, B. cereus used the mixed acid fermentation to ferment the different carbohydrates. The enterotoxin productions by this strain at μ = 0.2 h⁻¹ are highly influenced by the carbohydrates that do not involve any fermentative metabolism changes.     

The Effect of Calcium and Magnesium on the Activity of Bovicin HC5 and Nisin

Some Gram-positive bacteria produce small peptides (bacteriocins) that have antimicrobial activity, but many bacteria can become bacteriocin resistant. Bovicin HC5, a lantibiotic produced by Streptococcus bovis HC5, has the ability to inhibit nisin-resistant bacteria. Because nisin resistance has in many cases been correlated with an alteration of lipoteichoic acids or the polar head groups of membrane phospholipids, we decided to examine the effect of divalent cations on nisin and bovicin HC5 activity. Both bacteriocins catalyzed potassium efflux from S. bovis JB1, a non–bacteriocin-producing strain. The addition of large amounts (100 mM) of calcium or magnesium increased the ability of S. bovis JB1 to bind Congo red (an anionic dye) and counteracted bacteriocin-mediated potassium loss. Calcium was more effective than magnesium in decreasing nisin activity, but the reverse was observed with bovicin HC5. Nisin-resistant S. bovis JB1 cells bound three times as much Congo red as nisin-sensitive cells, and this result is consistent with the idea that changes in cell surface charge can be a mechanism of bacteriocin resistance. The nisin-resistant cells were less susceptible to bovicin HC5, but bovicin HC5 still caused a 50% depletion of intracellular potassium. These results indicate that nisin and bovicin HC5 react differently with the cell surfaces of Gram-positive bacteria. Proprietary or names are necessary to report factually on available data; however, the United States Department of Agriculture (USDA) neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

Effects of Chlorhexidine Diacetate on Ruminal Microorganisms

The objectives of this study were to examine the effects of chlorhexidine diacetate on growth and L-lactate production by Streptococcus bovis JB1 as well as the effects of this antimicrobial compound on the mixed ruminal microorganism fermentation. Addition of 1.8 μM chlorhexidine diacetate to glucose medium resulted in a lag in growth by S. bovis JB1, and growth was completely inhibited in the presence of 3.6, 9.0, and 18 μM chlorhexidine. When 6.2 μM chlorhexidine diacetate was added to glucose medium after 2 h of incubation, glucose utilization and L-lactate production by S. bovis JB1 were reduced. Phosphoenolpyruvate-dependent phosphorylation of ¹⁴C-glucose by toluene-treated cells of S. bovis JB1 was inhibited by increasing concentrations (1.8 to 18 μM) of chlorhexidine, whereas only the 18 μM concentration reduced the membrane potential (ΔΨ). Chlorhexidine diacetate was a potent inhibitor of L-lactate and methane production from glucose fermentation by mixed ruminal microorganisms. However, because chlorhexidine also decreased acetate and propionate concentrations and increased ammonia concentrations in mixed-culture incubations, this antimicrobial compound may have limited application as a ruminant feed additive. Received: 4 November 1997 / Accepted: 22 December 1997     

The effect of levofloxacin concentration on the development and maintenance of antibiotic-resistant clones of Escherichia coli in chemostat culture

A levofloxacin-sensitive strain of Escherichia coli (broth MIC: 0.0625 mg l⁻¹) was grown in carbon-limited chemostat culture for 316 h (D=0.294 h⁻¹). Hyperresistant strains isolated after 58 and 91 generations of culture retained a 16- to 47-fold increase in tolerance to levofloxacin during antibiotic-free serial batch and continuous culture (20 generations, glucose-limited, D=0.2 h⁻¹). Isolates differed from the original strain in their maximum growth rates in the presence and absence of subinhibitory levels of levofloxacin, protein-banding profiles, and resistance to a range of antibiotics. Competition between resistant isolates and the original sensitive strain was studied in glucose-limited chemostat cultures (D=0.2 h⁻¹). At levofloxacin concentrations less than 0.03 mg l⁻¹, the sensitive strain outcompeted resistant isolates and displaced them from the culture, whereas the reverse was true at higher concentrations. These results have clinical and environmental implications for those administering levofloxacin. Journal of Industrial Microbiology & Biotechnology (2002) 29, 155–162 doi:10.1038/sj.jim.7000295 Received 26 September 2001/ Accepted in revised form 13 June 2002     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.     

Effects of Thymol on Ruminal Microorganisms

Thymol (5-methyl-2-isopropylphenol) is a phenolic compound that is used to inhibit oral bacteria. Because little is known regarding the effects of this compound on ruminal microorganisms, the objective of this study was to determine the effects of thymol on growth and lactate production by the ruminal bacteria Streptococcus bovis JB1 and Selenomonas ruminantium HD4. In addition, the effect of thymol on the in vitro fermentation of glucose by mixed ruminal microorganisms was investigated. Neither 45 nor 90 μg/ml of thymol had any significant effect on growth or lactate production by S. bovis JB1, but 180 μg/ml of thymol completely inhibited growth and lactate production. In the case of S. ruminantium HD4, 45 μg/ml of thymol had little effect on growth and lactate production; however, 90 μg/ml of thymol completely inhibited growth of S. ruminantium HD4. Thymol also decreased glucose uptake by whole cells of both bacteria. When mixed ruminal microorganisms were incubated in medium that contained glucose, 400 μg/ml of thymol increased final pH and the acetate to propionate ratio and decreased concentrations of methane, acetate, propionate, and lactate. In conclusion, thymol was a potent inhibitor of glucose fermentation by S. bovis JB1 and S. ruminantium HD4. Even though thymol treatment decreased methane and lactate concentrations and increased final pH in mixed ruminal microorganism fermentations of glucose, concentrations of acetate and propionate were also reduced. Received: 13 May 2000 / Accepted: 14 June 2000     

Growth and protopectinase production of Geotrichum klebahnii in batch and continuous cultures with synthetic media

Geotrichum klebahnii ATCC 42397 produces a protopectinase (PPase-SE) with polygalacturonase (PGase) activity. The microorganism was aerobically cultivated in synthetic media. Glucose, fructose and xylose yielded the highest enzyme levels (10–11 PGase units ml⁻¹). Galacturonic acid repressed enzyme production and no growth was obtained with disaccharides and pectin. Specific enzyme activity obtained in an O₂-limited culture was similar to that found in nonlimited ones. A growth yield (Y _x/s) of 0.49 g of cell dry weight per gram of glucose consumed was obtained in a typical batch bioreactor culture. Enzyme production was growth associated, and no major products other than biomass and CO₂ were detected. The volumetric enzyme activity reached a maximum around D=0.3–0.4 h⁻¹ in glucose-limited continuous cultures. However, it varied strongly (together with microorganism morphology) even after retention times ≥8 at any D tested (0.035–0.44 h⁻¹) though the rest of the culture variables remained fairly constant. No correlation between morphology and enzyme activity could be obtained. Enzyme production was poor in urea- and vitamin-limited continuous cultures. In all cases, biomass and CO₂ accounted for ≅100% of carbon recovery though Y _x/s values were different. Journal of Industrial Microbiology & Biotechnology (2000) 25, 260–265. Received 20 April 2000/ Accepted in revised form 15 September 2000     

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     

Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose

In order to improve the production rate of l-lysine, a mutant of Corynebacterium glutamicum ATCC 21513 was cultivated in complex medium with gluconate and glucose as mixed carbon sources. In a batch culture, this strain was found to consume gluconate and glucose simultaneously. In continuous culture at dilution rates ranging from 0.2 h⁻¹ to 0.25 h⁻¹, the specific l-lysine production rate increased to 0.12 g g⁻¹ h⁻¹ from 0.1 g g⁻¹ h⁻¹, the rate obtained with glucose as the sole carbon source [Lee et al. (1995) Appl Microbiol Biotechnol 43:1019–1027]. It is notable that l-lysine production was observed at higher dilution rates than 0.4 h⁻¹, which was not observed when glucose was the sole carbon source. The positive effect of gluconate was confirmed in the shift of the carbon source from glucose to gluconate. The metabolic transition, which has been characterized by decreased l-lysine production at the higher glucose uptake rates, was not observed when gluconate was added. These results demonstrate that the utilization of gluconate as a secondary carbon source improves the maximum l-lysine production rate in the threonine-limited continuous culture, probably by relieving the limiting factors in the lysine synthesis rate such as NADPH supply and/or phosphoenolpyruvate availability. Received: 16 May 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997     

Continuous acetone–butanol–ethanol production by corn stalk immobilized cells

Corn stalk was used as a support to immobilize Clostridia beijerinckii ATCC 55025 in the fermentation process of acetone, butanol, and ethanol production. The effect of the dilution rate on solvent production was examined in a steady-state 20-day continuous flow operation. The maximum total solvent concentration of 8.99 g l⁻¹ was obtained at a dilution rate of 0.2 h⁻¹. Increasing the dilution rate between 0.2 and 1.0 h⁻¹ resulted in an increased solvent productivity, and the highest solvent productivity was obtained at 5.06 g l⁻¹ h⁻¹ with a dilution rate of 1 h⁻¹. The maximum solvent yield from glucose of 0.32 g g⁻¹ was observed at 0.25 h⁻¹. The cell adsorption and morphology change during the growth on corn stalk support were examined by the SEM.     

Continuous bio-catalytic conversion of sugar mixture to acetone–butanol–ethanol by immobilized <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> DSM 792

Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L⁻¹ was obtained at a dilution rate of 0.22 h⁻¹ with glucose as a substrate compared to 12.64 g L⁻¹ at 0.5 h⁻¹ dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L⁻¹ h⁻¹) was obtained at a dilution rate of 1.9 h⁻¹ with glucose as a substrate whereas solvent productivity (12.14 g L⁻¹ h⁻¹) was obtained at a dilution rate of 1.5 h⁻¹ with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries.     

Application of oscillation for efficiency improvement of continuous ethanol fermentation with Saccharomyces cerevisiae under very-high-gravity conditions

Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280 g l⁻¹ glucose was fed at the dilution rate of 0.04 h⁻¹, the average ethanol of 15.8% (v/v) and residual glucose of 1.5 g l⁻¹ were achieved under the oscillatory state, with an average ethanol productivity of 2.14 g h⁻¹ l⁻¹. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l⁻¹, with an ethanol productivity of 2.00 g h⁻¹ l⁻¹, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h⁻¹ to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l⁻¹, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h⁻¹ l⁻¹, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h⁻¹.     

Performance assessment of malolactic fermenting bacteria Oenococcus oeni and Lactobacillus brevis in continuous culture

The growth performance of malolactic fermenting bacteria Oenococcus oeni NCIMB 11648 and Lactobacillus brevis X₂ was assessed in continuous culture. O. oeni grew at a dilution rate range of 0.007 to 0.052 h⁻¹ in a mixture of 5:6 (g l⁻¹) of glucose/fructose at an optimal pH of 4.5, and L. brevis X₂ grew at 0.010 to 0.089 h⁻¹ in 10 g l⁻¹ glucose at an optimal pH of 5.5 in a simple and safe medium. The cell dry weight, substrate uptake and product formation were monitored, as well as growth kinetics, yield parameters and fermentation balances were also evaluated under pH control conditions. A comparison of growth characteristics of two strains was made, and this showed significantly different performance. O. oeni has lower maximum specific growth rate (μ_max=0.073 h⁻¹), lower maximum cell productivity (Q _x ^max=17.6 mg cell l⁻¹ h⁻¹), lower maximum biomass yield (Y _x/s ^max=7.93 g cell mol⁻¹ sugar) and higher maintenance coefficient (m _s=0.45 mmol⁻¹ sugar g⁻¹ cell h⁻¹) as compared with L. brevis X₂ (μ_max=0.110 h⁻¹; Q _x ^max=93.2 g⁻¹ cell mol⁻¹ glucose; Y _x/s ^max=22.3 g cell mol⁻¹ glucose; m _s=0.21 mmol⁻¹ glucose g⁻¹ cell h⁻¹). These data suggest a possible more productive strategy for their combined use in maturation of cider and wine.     

Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling

The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h⁻¹ and 1.0 h⁻¹ and glycerol input concentrations of 32 g l⁻¹ and 56 g l⁻¹. The near-to-optimum propanediol concentration of 26.5 g l⁻¹ (for 56 g l⁻¹ glycerol) was maintained up to a dilution rate of 0.5 h⁻¹ and then decreased while the propanediol productivity was highest at 0.7 h⁻¹. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling. By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling system, such as shear stress, were not involved. Received: 20 October 1997 / Received revision: 12 December 1997 / Accepted: 14 December 1997     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.