Regulation of plasmid-encoded lactose genes in different gram-negative enterobacteriaceae

The expression and regulation of a variety of plasmid-encoded lactose systems have been studied in different gram-negative Enterobacteriaceae (Escherichia coli K-12, Serratia marcescens, and Enterobacter liquefaciens). They are similarly expressed in all strains, and they are inducible and sensitive to catabolite repression. Urea, known to bring about a specific catabolite repression effect, has been chosen to study some regulatory aspects of these plasmid-encoded lactose systems. We have shown that the expression of all lactose systems is inhibited by urea but the extent of the inhibition is strain dependent. This indicates that the genetic background of the host might modify in certain instances the expression of plasmid-encoded genes.     

Ethanol production from lactose by extractive fermentation

Summary The suitability of extractive fermentation as a technique for the production of ethanol from lactose by Candida pseudotropicalis was examined as a potential improvement over conventional methods. A biocompatible solvent was selected through determination of the critical log P (octanol-water distribution coefficient) of the fermentation organism. Using Adol 85 NF, the selected solvent, extractive fed-batch and conventional fed-batch systems were operated for 160 h. The extractive system showed a 60% improvement in lactose consumption and ethanol production, as well as a 75% higher volumetric productivity.     

Acidogenic fermentation of lactose

Cheese whey is the main component of waste streams from cheese manufacturing plants. Whey is a high biochemical oxygen demand (BOD) effluent that must be reduced before the streams are sent to the sewer. It is proposed in this article that the production of methane by anaerobic fermentation would be the best use of this stream, especially for small plants. Single-stage fermentation of lactose, the main component of whey, results in a very low pH and a stalled process. Two-phase fermentation will eliminate this problem. The acidogenic stage of fermentation has been studied at pH of between 4 and 6.5. The nature of the main products of the reaction have been found to be pH dependent. Below a pH of 4.5 a gas (CO(2) and H(2)) is produced along with ethanol, acetate, and butyrate. Above a pH of 4.5 no gas was produced, and the liquid products included less ethanol and butyrate and more acetate. A separate study on the conditions for gas formation showed that if the pH dropped for a short time below 4.5 gases were formed at all subsequent pH. This would indicate a change in population distribution due to the period at a low pH. By assuming that the desired products from the acidogenic stage were butyrate, acetate, and no gases, the optimum pH range was found to be between 6.0 and 6.5.     

Introduction of the tac-promoter by lactose under fermentation conditions

The use of the expensive IPTG for induction of the tac-promoter is not practicable at the industrial scale because of the large amounts necessary for induction. We developed a system in which IPTG as inducer was replaced by lactose which induces the tac-promoter with the same efficiency. In E. coli CW3011 (pOF1.0) lactose is utilized as inducer and carbon source even in the presence of glucose, presumably as a consequence of the overproduction of lac-permease.     

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of acidogenesis kinetics

An assessment of the growth kinetics of acidogenic cells of Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a continuous stirred tank reactor under controlled conditions adopting a complex medium supplemented with lactose as carbon source to mimic cheese whey. The effects of acids (acetic and butyric), solvents (acetone, ethanol and butanol) and pH on the growth rate of acidogenic cells were assessed. The conversion process was characterized under steady-state conditions in terms of concentration of lactose, cells, acids, total organic carbon and pH. The growth kinetics was expressed by means of a multiple product inhibition and interacting model including a novel formulation to account for the role of pH. The model has the potential to predict microorganism growth rate under a broad interval of operating conditions, even those typical of solvents production.     

Preparation of lactose free milk by fermentation using immobilizedSaccharomyces fragilis

Summary Saccharomyces fragilis cells (40% w/v) were immobilized in 2% Ca-alginate and were used in a batch process for the removal of lactose from milk by fermentation. Immobilized cells (10 g) could completely desugarate 100 mL of milk in 3.5 h. The immobilized preparation was used repeatedly in 15 batches without decrease in the activity.     

Delayed fermentation of sucrose by certain haploid species ofSaccharomyces

Summary Several haploid species ofSaccharomyces andSchiz. octosporus were shown to ferment sucrose in Durham tubes after a delay of 3 to 4 weeks. Detailed studies were done with a strain ofS. rouxii. The delayed fermentation of sucrose was not caused by mutationselection or by inducible enzyme formation, since young glucose grown cells after drying, freezing, aging or autolysis contained an active sucrase. Cells pretreated by drying or freezing fermented sucrose nearly as fast as glucose. After autolysis, the sucrase ofS. rouxii is only present in the cell debris and not in the autolysate. The use of a heavy inoculum in the van Iterson-Kluyver fermentometer resulted in a slow, but non-delayed fermentation. Variation in the pH or sucrose concentration had little effect on the delayed fermentation. It is suggested that after sufficient aging of the cells, the cell wall permeability undergoes a rather abrupt change, allowing the sucrose to come in contact with the sucrase of the cells.     

Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH

Batch propionic acid fermentation of lactose by Propionibacterium acidipropionici were studied at various pH values ranging from 4.5 to 7.12. The optimum pH range for cell growth was between 6.0 and 7.1, where the specific growth rate was approximately 0.23 h(-1). The specific growth rate decreased with the pH in the acids have been identified as the two major fermentation products from lactose. The production of propionic acid was both growth and nongrowth associated, while acetic acid formation was closely associated with cell growth. The propionic acid yield increased with decreasing pH; It changed from approximately 33% (w/w) at pH 6.1-7.1 to approximately 63% at pH 4.5-5.0. In contrast, the acetic acid yield was not significantly affected by the pH; it remained within the range of 9%-12% at all pH values. Significant amounts of succinic and pyruvic acids were also formed during propionic acid fermentation of lactose. However, pyruvic acid was reconsumed and disappeared toward the end of the fermentation. The succinic acid yield generally decreased with the pH, from a high value of 17% at pH 7.0 to a low 8% at pH 5.0 Effects of growth nutrients present in yeast ex-tract on the fermentation were also studied. In general, the same trend of pH effects was found for fermentations with media containing 5 to 10 g/L yeast extract. However, More growth nutrients would be required for fermentations to be carried out efficienytly at acidic pH levels.     

Research perspectives and role of lactose uptake rate revealed by its study using 14C-labelled lactose in whey fermentation

The present investigation examines the effect of pH, temperature and cell concentration on lactose uptake rate, in relation with kinetics of whey fermentation using kefir and determines the optimum conditions of these parameters. Lactose uptake rate was measured by adding ¹⁴C-labelled lactose in whey. The results reveal the role of lactose uptake rate, being the main factor that affects the rate of fermentation, in contrast to the activity of the enzymes involved in lactose bioconversion process. Lactose uptake rate results discussion showed that mainly Ca²⁺ is responsible for the reduced whey fermentation rate in comparison with fermentations using synthetic media containing lactose. Likewise, the results draw up perspectives on whey fermentation research to improve whey fermentation rate. Those perspectives are research to remove Ca²⁺ from whey, the use of nano and microtubular biopolymers and promoters such as γ-alumina pellets and volcan foaming rock kissiris in order to accelerate whey fermentation.     

Regulation of lactose fermentation in group N streptococci

Group N streptococci, which have the lactose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) and phospho-beta-d-galactosidase (beta-Pgal), grew rapidly on lactose and converted more than 90% of the sugar to l-lactate. In contrast, Streptococcus lactis 7962, which does not have a beta-Pgal, grew slowly on lactose and converted only 15% of the sugar to l-lactate. With glucose and galactose, this strain had growth rates and fermentation patterns similar to those of other S. lactis strains, suggesting that the rapid and homolactic fermentation of lactose that is characteristic of group N streptococci is dependent upon a functional PEP-dependent PTS and the presence of beta-Pgal. Seventeen strains of group N streptococci were examined for the activator specificities of pyruvate kinase and lactate dehydrogenase. The properties of each enzyme from all the strains, including S. lactis 7962, were similar. Pyruvate kinase had a broad activator specificity, whereas activation of lactate dehydrogenase was specific for ketohexose diphosphate. All intermediates of lactose metabolism from the hexose phosphates to the triose phosphates activated pyruvate kinase. No activation was obtained with adenosine 5'-monophosphate. K and Mg were required for pyruvate kinase activity but could be replaced by NH(4) and Mn, respectively. Lactate dehydrogenase was activated equally by fructose-1,6-diphosphate and tagatose-1,6-diphosphate, the activation characteristics being pH dependent. The roles of pyruvate kinase and lactate dehydrogenase in the regulation of lactose fermentation by group N streptococci are discussed.     

Intrinsic fermentation kinetics of lactose in acidogenic biofilms

The intrinsic fermentation kinetics of lactose in acidogenic biofilms were investigated in situ in a continuous flow fermentor at 35 degrees C and pH 4.6. The external and internal mass transfer resistances to lactose molecules from bulk solution to inside the biofilms were experimentally minimized or eliminated in a thin biofilm and recycled medium. In a chemically defined culture medium, the immobilized acidogens converted lactose mainly to acetate and butyrate; the minor products included ethanol. propionate, lactate, and hydrogen. The utilization rate of lactose, as a function of lactose concentration in the fermentor, can be described by a Michaelis-Menten equation, as can the formation rates of acetate, butyrate, and ethanol. The production rates of propionate and lactate had a liner relationship with lactose concentration under the experimental conditions. The low pH (4.6) of culture medium could depress the formation of propionate, and intermediate which is most difficulty digested by acetogenic bacteria located in the second fermentor in a two-phase process. Production rate of acetate quickly reached a constant, and additional utilization of lactose produced more butyrate and other minor products. (c) 1993 John Wiley & Sons, Inc.     

Continuous ethanol fermentation of lactose by a recombinant flocculating Saccharomyces cerevisiae strain.

Alcohol fermentation of lactose was investigated using a recombinant flocculating Saccharomyces cerevisiae, expressing the LAC4 (coding for beta-galactosidase) and LAC12 (coding for lactose permease) genes of Kluyveromyces marxianus. Data on yeast fermentation and growth on a medium containing lactose as the sole carbon source are presented. In the range of studied lactose concentrations, total lactose consumption was observed with a conversion yield of ethanol close to the expected theoretical value. For the continuously operating bioreactor, an ethanol productivity of 11 g L(-1) h(-1) (corresponding to a feed lactose concentration of 50 g L(-1) and a dilution rate of 0.55 h(-1)) was obtained, which is 7 times larger than the continuous conventional systems. The system stability was confirmed by keeping it in operation for 6 months.     

Whey fermentation: on-line analysis of lactose and lactic acid by FTIR spectroscopy

Summary The batch fermentation of whey permeate by Lactobacillus helveticus 8652, was monitored on-line by Fourier transform infrared (FTIR) spectroscopy. Substrate (lactose) and product (lactic acid) levels were measured over a period of 47 h. A method for the quantitative analysis of the two components was first established. Fifteen standard solutions containing both lactose and lactic acid were used. The method developed was tested using validation samples of known composition. Mean errors of 1.1% and 0.9% were attained in the measurement of lactose and lactic acid respectively. Sample analysis is fast (approx. 3 min), simple and almost completely automated. The results obtained with FTIR spectroscopy compared favourably with samples analysed off-line using enzyme kits. Offprint requests to: P. Fairbrother     

Identification of lactose fermentation plasmids of streptococcal dairy starter strains by Southern hybridization

Twenty-two strains of Streptococcus cremoris , seven strains of Streptococcus lactis and three strains of Streptococcus lactis subsp. diacetilactis, each with a different plasmid complement, were isolated from a starter culture used in a Finnish dairy plant. By using DNA-DNA hybridization, with cloned 6-P-ß-galactosidase gene of the Strep, lactis plasmid pLP712 as a probe, the lactose fermentation genes were located, in each strain, in the large ( 30 MD) plasmid.     

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO₂ of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.     

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of energetics and product yields of the acidogenesis

An assessment of both the growth and the metabolism of acidogenic cells Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a CSTR under controlled pH conditions. Cultures were carried out using a semi-synthetic medium supplemented with lactose as carbon source. Acids and solvents, that represent products of the ABE process, have been purposely added in controlled amounts to the culture medium to investigate their effects on the product yields. The mass fractional yield of biomass and products were expressed as a function of the specific growth rate taking into account the Pirt model. The maximum ATP yield and the maintenance resulted 29.1 g(DM)/mol(ATP) and 0.012 mol(ATP)/g(DM)h, respectively. Quantitative features of the C. acetobutylicum growth model were in good agreement with experimental results. The model proposes as a tool to estimate the mass fractional yield even for fermentations carried out under conditions typical of the solventogenesis.