Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by <Emphasis Type="Italic">Lactobacillus bifermentans</Emphasis>

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, and incubation period) and culture medium composition (mineral salts, carbon source, and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. For optimal growth, the strain required Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g., ammonium citrate). The bacterium had no requirement for sodium acetate for either growth or production of isomerases. The production rate of enzymes was increased when metal ions were added, primarily manganese (2.5 mM). The text was submitted by the authors in English.     

Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Analysis of <Emphasis Type="Italic">ldh</Emphasis> genes in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> BL23: role on lactic acid production

Lactobacillus casei is a lactic acid bacterium that produces L-lactate as the main product of sugar fermentation via L-lactate dehydrogenase (Ldh1) activity. In addition, small amounts of the D-lactate isomer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 draft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the global lactate production in this organism, individual, as well as double mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their single mutation or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A Deltaldh1 mutant displayed an increased production of D-lactate, which was probably synthesized via the activity of HicD, as it was abolished in a Deltaldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic acid.     

Enhanced production of lactic acid by an adapted strain of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactobacillus delbrueckii subsp. lactis strains were developed having increased activity, by gradually acclimatizing the bacteria to acidic conditions over repeated batch culture. Cells from one batch culture were used as the inoculum for the subsequent batch culture and thereby an adapted strain of Lactobacillus was obtained showing improved lactic acid productivity, cell growth and total glucose utilization. Furthermore, the acclimatized cells used significantly less nitrogen for a given level of lactic acid production, which is significant from an industrial point of view. The developed procedure decreases fermentation time and nutrient use, leading to reduced operation costs, while providing a lactic acid yield superior to previously reported methods.     

Improved immobilization of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> ATCC 7469 in polyacrylamide gel,preventing cell leakage during lactic acid fermentation

A new procedure for improved immobilization of Lactobacillus rhamnosus ATCC 7469, producing solely l(+)-lactic acid, in polyacrylamide was developed. A series of gels with varied ingredients concentrations and order of addition was prepared and were tested in batch and repeat-batch processes. Our results revealed that the crucial step for successful immobilization was the initial incubation of the cells in pure 10% AA that leads to improved entrapment in the polyacrylamide gel. In contrast, all gels derived from previously prepared stock AA/MBAA released high amount of cells and free biomass was formed. The most efficient immobilization was achieved using gel, containing L. rhamnosus, incubated in 10% AA (acrylamide) and with 1% MBAA (N,N′-methylene-bis-acrylamide) added. This gel possessed optimal permeation characteristics and at the same time, the cells were completely retained in the polymer lattice (0.03 g free biomass/l at 48 h of the batch process). In addition, it yielded highly concentrated lactic acid: the conversion ratio was about 85% without pH-control for initial lactose concentrations of up to 30 g/l. A series of additional immobilization experiments showed the potential of physicochemical interactions between the monomers of acrylamide and the cell surface of L. rhamnosus.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Degradation of ethylbenzene by free and immobilized <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens-</Emphasis>CS2

Pseudomonas fluorescens-CS2 metabolized ethylbenzene as the sole source of carbon and energy. The involvement of catechol as the hydroxylated intermediate during the biodegradation of ethylbenzene was established by TLC, HPLC and enzyme analysis. The specific activity of Catechol 2,3-dioxygenase in the cell free extracts of P. fluorescens-CS2 was determined to be 0.428 μmoles min⁻¹ mg⁻¹ protein. An aqueous-organic, Two-Phase Batch Culture System (TPBCS) was developed to overcome inhibition due to higher substrate concentrations. In TPBCS, P. fluorescens-CS2 demonstrated ethylbenzene utilization up to 50 mM without substrate inhibition on inclusion of n-decanol as the second phase. The rate of ethylbenzene metabolism in TPBCS was found enhance by fivefold in comparison with single phase system. Alternatively the alginate, agar and polyacrylamide matrix immobilized P. fluorescens-CS2 cells efficiently degraded ethylebenzene with enhanced efficiency compared to free cell cultures in single and two-phase systems. The cells entrapped in ployacrylamide and alginate were found to be stable and degradation efficient for a period of 42 days where as agar-entrapped P. fluorescens was stable and efficient a period of 36 days. This demonstrates that alginate and polyacrylamide matrices are more promising as compared to agar for cell immobilization.     

Production of high fructose syrup from <Emphasis Type="Italic">Asparagus</Emphasis> inulin using immobilized exoinulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K _m values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E _a) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co²⁺ and Mn²⁺ enhanced the activity, whereas Hg²⁺ and Ag²⁺ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.     

Decolorization of reactive dyes by <Emphasis Type="Italic">Clostridium bifermentans</Emphasis> SL186 isolated from contaminated soil

Decolorization of textile reactive azo dyes by a strain of bacteria (SL186) isolated from a contaminated site was investigated. SL186 was identified as Clostridium bifermentans by phenotypic characterization and 16S rDNA sequence comparison. Under anaerobic conditions, SL186 had decolorized the dyes Reactive Red 3B-A, Reactive Black 5, and Reactive Yellow 3G-P by over 90% after 36 h post-inoculation. The bacterium retained decolorizing activity over a wide range of pH values (6–12), with peak activity at pH 10. Additionally, SL186 decolorized a relatively high concentration of Reactive Red 3B-A dye (1,000 ppm) by over 80% and raw industrial effluent effectively. The addition of glucose increased the decolorization rate a little. Spectrophotometric analyses of the reactive dyes showed no distinct peak indicating aromatic amines. However, a new peak was detected between 300 and 450 nm from the decolorized raw industrial effluent. These results suggest that C. bifermentans SL186 is a suitable bacterium for the biological processing of dye-containing wastewater.     

Exopolysaccharide production of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> BCRC 14759 and <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> BCRC 14615

In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract of both test organisms. This indicates their possible involvement in the biosynthesis of EPS.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Optimizing emulsan production of <Emphasis Type="Italic">A. venetianus</Emphasis> RAG-1 using response surface methodology

Statistical experimental design was used to optimize medium constituents for emulsan production by Acinetobacter venetianus RAG-1 in batch cultivation. The factors affecting emulsan production were screened by a two-level factorial design, and the optimal concentration of medium constituents for emulsan production were determined by the method of steepest path ascent and central composite experimental design. Experimental results showed that the optimal medium constituents were 9.16 g/L ethanol, 8.2 g/L KH₂PO₄, 23.32 g/L K₂HPO₄, 5.77 g/L (NH₄)₂SO₄ and 0.354 g/L MgSO₄•7H₂O. Under this optimal composition, the predicted emulsan production was 72.198 mg/L, and experimental value was 73.312 mg/L for 80 h culture in the shake flasks, and the emulsan yield by A. venetianus RAG-1 was enhanced nearly 1.48-fold (from 49.5 to 73.312 mg/L). Based on the results, we identify the optimal medium constituents for emulsan production and could take advantage of strategy for scale up the fermentation of emulsan production.     

Optimization of medium composition for actinomycin X2 production by <Emphasis Type="Italic">Streptomyces </Emphasis><Emphasis Type="Italic">spp</Emphasis> JAU4234 using response surface methodology

The effects of cultivation medium compositions including soybean meal, peptone, soybean oil and cornstarch for actinomycin X2 production by Streptomyces spp JAU4234 were accessed by using response surface methodology. The 2(4) full factorial designs and the paths of steepest ascent were effective in searching for the major factors of actinomycin X2 production. In this study, cornstarch and soybean oil showed negative effect on actinomycin X2 production based on the first-order regression coefficients derived from MINITAB software. Subsequently, a central composite design for optimization was further investigated. Preliminary studies showed that soybean meal and peptone were believed to be the major factors for actinomycin X2 production. Estimated optimum compositions for the production of actionmycin X2 were as follows (g/l): soybean meal 21.65 and peptone 9.41, and result in a maximum actionmycin X2 production of 617.4 mg/l. This value was closed to the 612 mg/l actionmycin X2 production from actual experimental observations. The yield of actionmycin X2 was increased by 36.9% by culturing the strain Streptomyces spp JAU4234 in the nutritionally optimized fermentation medium.     

Probiotic properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains isolated from porcine gastrointestinal tract

One strain of Lactobacillus salivarius, two strains of Lactobacillus reuteri and Lactobacillus amylovorus, and two strains of Bifidobacterium thermacidophilum with antagonistic effect against Clostridium perfringens were isolated from porcine gastrointestinal tract. Isolates were assayed for their ability to survive in synthetic gastric juice at pH 2.5 and were examined for their ability to grow on agar plate containing porcine bile extract. There was a large variation in the survival of the isolates in gastric juice and growth in the medium containing 0.3% (w/v) bile. L. salivarius G11 and L. amylovorus S6 adhered to the HT-29 epithelial cell line. Cell-free supernatant of L. amylovorus S6 showed higher antagonistic activity as effective as the antibiotics such as neomycin, chlortetracycline, and oxytetracycline against bacterial pathogens including C. perfringens, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Edwardsiella tarda, and Aeromonas salmonicida subsp. salmonicida.     

Enhanced production of a thermostable mannanase by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> on various membranes

Cells of the thermophilic Bacillus subtilis WY34 were immobilized on various formaldehyde-activated polymer membranes and the immobilized cells were used for the production of thermostable mannanase in flasks. The results showed that polyethersulfone membranes (PES) and nylon-6 membranes were the most suitable supports for cell immobilization to produce the mannanase. Moreover, PES and nylon-6 membranes immobilized cells provided 1.78- and 1.74-fold higher mannanase activity compared to the control after 4 days of cultivation, respectively. The immobilized cells on PES and nylon-6 membranes had good stability and retained 131.5 and 114.3% of ability of enzyme production even after six cycles of repeated batch fermentation, respectively. Active cell growth was observed by scanning electron microscopy (SEM) after 16 days (four cycles) repeated batch cultivation. Therefore, the membrane-immobilized cells of B. subtilis WY34 can be proposed as an effective biocatalyst for repeated usage for production of the thermostable mannanase.     

Kinetic Modelling of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> ssp. <Emphasis Type="Italic">rhamnosus</Emphasis> Growth and Lactic Acid Production in Batch Cultures Under Various Medium Conditions

Enrichment of medium with yeast extract and tryptone increased growth and lactic acid production in batch cultures of Lactobacillus casei ssp. rhamnosus. A reliable kinetic model that explicitly expresses the strong relationship between microbial growth, lactic acid production and medium enrichment is provided and validated using experimental data obtained with six different medium compositions. Revisions requested 2 February 2005 and 26 July 2005; Revisions received 25 July 2005 and 9 September 2005     

An efficient process for production and purification of hyaluronic acid from <Emphasis Type="Italic">Streptococcus </Emphasis><Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5–6 g hyaluronic acid/l after 24–28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of ∼4 × 10⁶ Da with less than 0.1% protein.     

Influence of immobilization parameters on growth and lactic acid production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> co-immobilized in calcium alginate gel beads

Streptococcus thermophilusand Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1–1.5M) and alginate (1–2<><>, w/v) concentrations. Highest lactic acid production was 35 g l₁ when both bacteria were in high viscosity beads (1<><>, w/v alginate) hardened in 0.1 M CaCl₂ .The gel bead composition affected size and distribution of entrapped lactic acid bacteria.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.