Lactose Medium Research Articles

Growth and ?-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10-12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4-8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in 'substrate-accelerated death'. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO 2 . Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol.

Construction of Kluyveromyces lactis killer strains defective in growth on lactic acid as a silage additive

Killer strain of Kluyveromyces lactis was constructed to prevent the aerobic deterioration of silage by disruption of KlPCK1 gene encoding phosphoenolpyruvate carboxykinase (PEPCK). The disruptants are defective in their ability of growth on lactic acid as a sole carbon source. The PEPCK activity was also deficient in the disruptants. The growth rate on lactose medium and the killing activity were equal to those of the parental strain. © Rapid Science Ltd. 1998

Molecular cloning and sequencing of two phospho-beta-galactosidase I and II genes of Lactobacillus gasseri JCM1031 isolated from human intestine.

Lactobacillus (Lb.) gasseri JCM1031, which is classified into the B1 subgroup of the Lb. acidophilus group of lactic acid bacteria, characteristically produces two different phospho-beta-galactosidases (P-beta-gal) I and II in the same cytosol as reported in our previous papers [Biosci. Biotech. Biochem., 60, 139-141, 708-710 (1996)]. To clarify the functional and genetic properties of the two enzymes, the structural genes of P-beta-gal I and II were cloned and sequenced. The structural gene of P-beta-gal I had 1,446 bp, encoding a polypeptide of 482 amino acid residues. The structural gene of P-beta-gal II had 1,473 bp, encoding a polypeptide of 491 amino acid residues. The deduced relative molecular masses of 55,188 and 56,243 agreed well with the previous value obtained from the purified P-beta-gal I and II protein, respectively. Multiple alignment of the protein sequence of P-beta-gal I and II with those of P-beta-gals from 5 microorganisms had 30-35% identity on the amino acid level, but those with phospho-beta-glucosidases from 5 microorganisms had the relatively high identity of about 50%. Considering that this strain grows on lactose medium and shows no beta-galactosidase activity, and that purified P-beta-gal I and II can obviously hydrolyze o-nitrophenyl-beta-D-galactopyranoside 6-phosphate (substrate), and also the conservation of a cysteine residue in the molecule, the P-beta-gal I and II were each confirmed as a novel P-beta-gal enzyme.

CAMP receptor protein–cAMP plays a crucial role in glucose–lactose diauxie by activating the major glucose transporter gene in Escherichia coli

The inhibition of beta-galactosidase expression in a medium containing both glucose and lactose is a typical example of the glucose effect in Escherichia coli. We studied the glucose effect in the lacL8UV5 promoter mutant, which is independent of cAMP and cAMP receptor protein (CRP). A strong inhibition of beta-galactosidase expression by glucose and a diauxic growth were observed when the lacL8UV5 cells were grown on a glucose-lactose medium. The addition of isopropyl beta-D-thiogalactoside to the culture medium eliminated the glucose effect. Disruption of the crr gene or overproduction of LacY also eliminated the glucose effect. These results are fully consistent with our previous finding that the glucose effect in wild-type cells growing in a glucose-lactose medium is not due to the reduction of CRP-cAMP levels but is due to the inducer exclusion. We found that the glucose effect in the lacL8UV5 cells was no longer observed when either the crp or the cya gene was disrupted. Evidence suggested that CRP-cAMP may not enhance directly the lac repressor action in vivo. Northern blot analysis revealed that the mRNA for ptsG, a major glucose transporter gene, was markedly reduced in a delta crp or delta cya background. The constitutive expression of the ptsG gene by the introduction of a multicopy plasmid restored the glucose effect in delta cya or delta crp cells. We conclude that CRP-cAMP plays a crucial role in inducer exclusion, which is responsible for the glucose-lactose diauxie, by activating the expression of the ptsG gene.

Production of γ-Linolenic Acid by the Fungus Cunninghamella echinulata CCRC 31840

The effect of various carbon and nitrogen sources and carbon/nitrogen (C :N) ratios on the γ-linolenic acid (GLA) production by Cunninghamella echinulata CCRC 31840 was examined in shake cultures. On the same weight base, soluble starch was the most preferred carbon source for the growth of this fungal strain and also yielded the largest amount of GLA per unit volume. In the lactose medium, growth of C. echinulata CCRC 31840 was very poor but the most unsaturated lipid was produced. Ammonium nitrate and urea were the better nitrogen sources for growth than potassium nitrate, ammonium chloride, and ammonium sulfate. C. echinulata CCRC 31840 grown on KNO 3 acquired the richest lipid content (35.4%) ; however, the content of GLA in the lipid (7.9%) was much lower than that grown on urea (25.7%). An initial C :N ratio of approximately 33-48.5 favored lipid accumulation in biomass without affecting the GLA content of the lipid. A maximum GLA yield of 964 mg/L was obtained after 5 days of incubation at 20 °C in a medium containing 10% soluble starch, 0.5% yeast extract, 0.11% NH 4 NO 3 , 0.1% KH 2 PO 4 , and 0.025% MgSO 4 .7H 2 O.

Use of Differential Rumen Fluid-Based Carbohydrate Agar Media for Culturing Lactose-Selected Cecal Bacteria from Chickens

A rumen fluid-based differential carbohydrate agar medium for enumerating chicken cecal carbohydrate-utilizing bacteria was tested. Eleven bacteria isolated from a continuous-flow culture that had been seeded with chicken cecal contents were inoculated on fifteen different media in an anaerobic environment. These media included various levels of rumen fluid (0%, 5%, 10%, 16%) and carbohydrate (glucose [GLU], galactose [GAL], lactose[LAC]). Viable cell recovery (colony-forming units [CFU]/direct cell counts × 100%) was higher for 16% rumen fluid-supplemented carbohydrate media than the lower concentrations of rumen fluid-supplemented carbohydrate media. In addition, average viable cell recovery of all 11 bacteria on differential carbohydrate media were significantly (P < 0.05) higher in galactose and lactose media than on the glucose media. Data indicated that 16% rumen-fluid based media can be used to enumerate anaerobic, lactose-fermenting subgroups of chicken cecal bacteria.

Construction of Lactose-Utilizing Xanthomonas campestris with a Mini-Mu Derivative

Xanthomonas campestris is not able to grow in lactose media. The lactose operon from Escherichia coli as part of a mini-Mu phage was integrated at random sites in the chromosome of this bacterium. Clones expressing (beta)-galactosidase were selected. The resulting strain X. campestris 204, is suitable for production of xanthan gum directly from lactose.

Continuous semi‐solid cultivation for the production of cellulase by Trichoderma reesei mutants using a polyurethane foam carrier and a liquid medium

AbstractThe production of cellulase was investigated in semi‐solid state culture using the immobilized mycelium of Trichoderma reesei mutants on polyurethane foam impregnated with lactose medium. An extremely high value of about 2.6 FPU/ml was reached after the cultivation of T. reesei D‐78085 on a 0.5% lactose medium in continuous culture at a pH medium of 4.0 when a bioreactor with vertical polyurethane foam plates was used. The enzyme yield on lactose was 520 FPU/g of lactose metabolized in comparison with 160 FPU/g using a stirred tank bioreactor.

Improved regeneration response of creeping bentgrass and japonica rice by maltose and lactose

The effect of alternative carbohydrate sources to sucrose for plant regeneration from long-term cell cultures of creeping bentgrass (Agrostis palustris Huds.cv.Penncross) and japonica rice (Oryza sativa L.cv.Nipponbare) was studied. Both maltose and lactose supported a higher degree of regeneration compared to sucrose; in 8-and 19-month-old cultures of creeping bentgrass, the frequencies of regenerating calli remained at 76–93% and the numbers of plants regenerated were 8 to 36-fold higher. In 35-month-old cultures of japonica rice, 2–4% of the calli were capable of regeneration on maltose and lactose media. These results indicate that loss of plant regeneration in long-term cultures is caused, at least in part, by specific cultural conditions and not by genetic changes.

Isolation and characterization of mutants with impaired regulation of rpsA, the gene encoding ribosomal protein S1 of Escherichia coli.

In order to select mutants that would help to characterize the post-transcriptional regulation of rpsA, we constructed a strain in which the growth rate on lactose minimal medium is determined by the amount of an rpsA-lacZ' alpha-fragment fusion protein produced, even when this is encoded by a high-copy-number plasmid. In the parental strain, synthesis of the fusion protein is repressed by a wild-type rpsA gene, present on a compatible plasmid. Twenty-eight spontaneous and independent mutants, all of them mapping in the rpsA leader region, were isolated as strains that showed higher growth rates, on lactose medium, due to increased synthesis of the rpsA-lacZ' fusion protein. Among these mutants only three sequence changes were found, mapping 9, 10 and 27 bases upstream of the rpsA start codon. At both the -9 and -10 positions an A to G transition and at -27 a C to G transversion all resulted in a sequence with better complementarity to the 3' end of 16S rRNA. We also isolated two mutations mapping in the plasmid-encoded rpsA structural gene: an ochre nonsense mutation in codon 15 of the rpsA gene and a frameshift mutation, deleting the T residue at position +1186. To facilitate the in vitro assay of alpha-fragment activity we also constructed a strain that overproduces the alpha-acceptor fragment four-fold relative to a strain that is diploid for this lacZ delta M15 allele.

The physiology of Lactococcus lactis subsp. lactis biovar. diacetylactis immobilized in hollow-fibre bioreactors: glucose, lactose and citrate metabolism at high cell densities

Lactococcus lactis subsp. lactis biovar. diacetylactis was selected to study the physiological influences of immobilization and growth to high cell densities. Cells were cultivated on glucose or lactose medium in the presence and absence of citrate. With excess glucose the cells produced mainly lactate as the fermentation product (homofermentative) providing that not all of the substrate was consumed. The population so cultivated was exposed to extreme gradients of pH and lactate concentrations. When the glucose concentration was reduced the population showed a mixed product profile with half of the glucose being fermented to lactate, the remainder to formate, acetate, ethanol and 2,3-butanediol. Inclusion of citrate in the medium shifted the population to homofermentation, with respect to the amount of glucose or lactose consumed. The citrate was metabolized via the pyruvate-formate lyase and α-acetolactate synthase routes. The pH of the medium was shown to strongly influence the product profile from citrate, presumably by affecting the activity of the key enzymes of pyruvate metabolism. The lactococci immobilized at high cell densities show product profiles typical of carbohydrate limitation at low dilution rates.

Construction of stable lactose-utilizing Xanthomonas campestris by chromosomal integration of cloned lac genes using filamentous phage ?Lf DNA

Previously, we constructed a lactose-utilizing strain of Xanthomonas campestris, Xc17 (pKMϕLT), by cloning lacZY genes with the RK2-derived vector pLAFR1. In this study, the narrow-host-range, β-galactosidase expression plasmid pKMϕ was fused with an integration vector pS19 to form pSFϕ14. Following insertion into Xc17, pSFϕ14 was integrated into the host chromosome. The integration function was provided by the 0.85-kb EcoRI-PstI fragment from the filamentous phage ϕLf. The integration caused no adverse effect to the cells and was stable for at least 66 generations without selection. The engineered strain, Xc17::pSFϕ14, was able to grow as well and produce as much xanthan gum in lactose medium as the wild-type cells did in glucose medium, and the Xc17(pKMϕLT) in lactose medium. Therefore, Xc17::pSϕF14 is potentially useful for xanthan production by direct use of whey lactose as the fermentation substrate. This study has advanced one more step our efforts to contruct lactose-utilizing X. campestris and confirmed the feasibility of using pS19 as an integration vector.

Use of whey for production of exocellular polysaccharide by a mutant strain of Xanthomonas campestris.

Growth and kinetics of the production of exocellular polysaccharide was studied in a mutant strain of Xanthomonas campestris lac+ during cultivation in a submerged culture in a medium containing whey. The maximum production of the polymer was observed at the initial stage of the stationary growth phase of the culture. The mean production yield was about 1.4%. The results were comparable with those obtained during cultivation on a lactose medium.

Production of Propionibacterium freudenreichii subsp. shermanii in whey-based media

Diluted cheese whey permeate supplemented with FACMP (Fermented Ammoniated Condensed Milk Permeate) was used as a culture medium for the production of Propionibacterium freudenreichii subsp. shermanii. FACMP was obtained through a mixed fermentation of milk permeate by Lactobacillus helveticus and Streptococcus thermophilus under pH control with gaseous ammonia. Uptake of carbon substrates (lactose and lactic acid) and production of organic acids (propionic, acetic, pyruvic) were followed by HPLC. Addition of yeast extract and KH2PO4 increased the growth rate; a cell population of 1.3 × 1010 colony forming units/ml was obtained within 48 h (3% v/v inoculum). Replacing FACMP by commercial sodium lactate or full strength whey permeate was investigated. Overall, the lactose medium (whey) was less efficient than the lactate supplemented media (FACMP, sodium lactate) in promoting bacterial growth and acid production. The use of FACMP instead of commercially available synthetic lactate improved the bacterial growth of P. shermanii by 25%.

An effective method for culturing pollen tubes of potato

In vitro culture of pollen tubes is a convenient way to distinguish inviable pollen from stylar incompatibilities which prevent pollen tube growthin vivo. This usefulness ofin vitro pollen tube culture depends on how successful the technique is in promoting optimal pollen tube growth. Experiments were conducted to compare the effects of the type of sugar and method of aeration of cultures ofSolanum species (potato) pollen. Lactose was superior to sucrose in promoting pollen germination and pollen tube growth on hanging drop cultures. Germination and growth were greatly enhanced when pollen was cultured in actively aerated lactose medium. Since pollen growth comparable to that occurringin vivo was observed using lactose medium with active aeration, this technique may be useful in various types of research related to reproductive biology.

Salt stress on growth and acid production of Lactobacillus helveticus strain milano

The effect of the addition of chloramphenicol and various salts on growth and acid production of Lactobacillus helveticus strain milano has been investigated using lactose medium. Injection of chloramphenicol induced uncoupling of growth and acid production. Addition of high salt concentrations also resulted in growth inhibition whereas product formation continued. NaCl and CaCl2 were more inhibitory to the growth of Lact. helveticus strain milano than KCl.

Utilization of citrate byLeuconostoc mesenteroides subsp.cremoris in continuous culture

Leuconostoc mesenteroides subsp.cremoris was grown in continuous culture in lactose medium with varying citrate concentrations. All citrate (10, 25, 50 and 75 mMol/l) was used and lactose consumption increased with increasing initial citrate concentrations correlate with an increase of dry cell weight. Citrate lead to an increase of acetate and could be a source of ATPvia acetate kinase pathway. For each steady state, YATP values were calculated and were twice greater than the generally accepted value of 10.5. The maintenance energy was calculated it was constant for lactose (2.5 mMol/l.h.) and increased for citrate suggesting a greater requirement of energy for citrate utilization.

Utilization of sugars by Lactobacillus acidophilus strains

Utilization of various carbohydrates viz., glucose, fructose, sucrose, lactose and galactose by Lactobacillus acidophilus strains was investigated in Lactobacillus Selection Broth. Maximum viable counts, acid production and sugar utilization by different test strains were in the order: glucose ⩾ fructose > sucrose ⩾ lactose > galactose. The generation time of the tested strains was shorter in glucose medium as compared to sucrose or lactose medium.

Influence of sugar source (lactose, glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose + galactose with final concentrations of up to 18.8g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose + galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose + galactose when aeration rate tends toward zero. Corresponding, the metabolism is more oxidative in the former medium, yielding mainly acetate as product.

Ethanol production from lactose by coculture of Kluyveromyces fragilis and Zymomonas mobilis

Ethanol production from lactose was studied using an adapted initial inoculum of Kluyveromyces fragilis NRRL 665 in monoculture and coculture with Zymomonas mobilis NRRL B 4286. The monoculture of adapted K. fragilis produced more ethanol than the non-adapted cultures. K. fragilis produced 52.98 g/ l ethanol in monoculture, and with Z. mobilis in coculture produced 61.29 g/ l ethanol from 250 g/ l lactose medium. The percentage of theoretical yield was found to be higher in coculture than in monoculture. With increasing substrate concentration from 100 g/ l to 250 g/ l, the parameters related to growth and sugar uptake were affected in both cases.