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Lactococcus lactis IO-1 was able to use xylose as a carbon source for nisin Z production; the yield based on sugar consumption was about 20% superior to that made with glucose under the same fermentation conditions. The optimal conditions for nisin Z production were with 4% xylose at pH 6.0 and 37°C. Addition of 0.1 M CaCl2 increased nisin Z production specifically, but not cell growth, acid production, or xylose consumption, and resulted in the maximum nisin Z activity of about 1.5 times that without CaCl2.

Bacteriocin ISK-1 is a proteinaceous inhibitory substance produced by Pediococcus sp. ISK-1 isolated from well-aged Nukadoko. Bacteriocin ISK-1 was purified by acid treatment, ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase HPLC from the culture supernatant of Pediococcus sp. ISK-1. Purification of bacteriocin ISK-1 resulted in a 30-fold increase in the specific activity and the recovery was 17%. Molecular mass of bacteriocin ISK-1 measured by fast atom bombardment-mass spectrometry was 2,960. The amino acid composition analysis of bacteriocin ISK-1 showed that it contained unusual amino acids such as lanthionine and/or 3-methyllanthionine, which is a characteristic of lantibiotics. The N-terminal amino acid sequence analysis indicated the first seven N-terminal amino acid residues as NH2-K-K-K-S-G-V-I. The primary sequence showed significant similarity to the lantibiotics lacticin 481 from Lactococcus lactis and variacin from Micrococcus varians, which suggests that bacteriocin ISK-1 is a novel lantibiotic belonging to a lacticin-481 type.

A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l(-1) h(-1) using a cell concentration of 15 g l(-1), 30( degrees )C, pH 5.5 and a dilution rate of 1.24 h(-1). Adding 10 g l(-1) YE and 2 g l(-1) polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.

The feasibility of utilizing enzymatic hydrolysates of separator sludge, a palm oil mill effluent, and sago starch in the acetone–butanol–ethanol fermentation by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) was investigated. The sludge hydrolysate was used as a growth medium substitute, a fermentation substrate, and as a source of nitrogen and micronutrients. Butanol produced, 3.50 g l−1, was the same when the sludge hydrolysate was used as a second (refreshing) growth medium. As a substrate, initial nitrogen gas sparging shortened the lag phase which then enhanced butanol production. Fermentation without pH control enhanced butanol productivity whereas controlled fermentation at pH 5.5 and 5.8 progressively favored acid production while redox dyes, methyl and benzyl viologen, neutral red and methylene blue, had no effect on solvent production. As a source of nitrogen and micronutrients in sago starch hydrolysate, the final butanol concentration, 10.4 g l−1, was comparable to that in glucose and sago starch hydrolysate media supplemented with tryptone–yeast–acetate (TYA) medium components medium, although productivity was low. Cell growth, butanol and total solvent productivity in TYA-supplemented sago starch hydrolysate and glucose media were the same.

Aims: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. Methods and Results: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca2+ (CaCO3 or CaCl2). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6·0, whereas the highest cell growth was obtained at pH 7·0. Conclusions: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca2+ and pH) influenced the bacteriocin production. Significance and Impact of the Study: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.

We have cloned and characterized the dnaK operon of Tetragenococcus halophila JCM5888. Nucleotide sequence analysis of cloned fragments showed that the dnaK operon consists of four open reading frames with the organization hrcA-grpE-dnaK-dnaJ. Two regulatory CIRCE (Controlling Inverted Repeat of Chaperone Expression) elements were identified in the region up-stream of hrcA. The T. halophila dnaK encoded a protein of 618 amino acids with a calculated molecular mass of 67 kDa. The deduced amino acid sequence of T. halophila DnaK showed high similarities with those of the corresponding DnaK homologues of Lactococcus lactis, Lactobacillus sakei and Bacillus subtilis. Using a pET expression system, the T. halophila DnaK was overexpressed in Escherichia coli and the purified DnaK was found to exhibit ATPase and refolding activities. Northern hybridization analysis revealed that the transcription of the dnaK gene was induced by heat shock, and several transcripts were detected including a tetra-cistronic mRNA with a maximum size of 4.9-kb which corresponds to the transcript of the complete dnaK operon. The amount of dnaK transcripts increased about 3.5-fold at high NaCl concentration of 3-4 M, but not at the same KCl concentrations. These results suggest that the cloned DnaK acts as a functional molecular chaperone and plays an important role in salinity adaptation.

The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila was cloned by a PCR-based method. The molecular masses of GroES and GroEL proteins were calculated to be 10,153 and 56,893 Da, respectively. The amount of groESL mRNA was increased 3.8-fold by heat shock (45 degrees C), and 4-fold by high NaCl (3-4 M). The Bacillus subtilis sigmaA-like constitutive promoter existed in front of groES, and was used under both normal and stress (heat shock and high salinity) conditions.

Enterococcus faecium WHE 81, isolated from cheese, has been reported to produce a bacteriocin called "enterocin 81" [J. Appl. Microbiol. 85 (1998) 521.]. Purification of "enterocin 81" was carried out using ammonium sulfate precipitation, desalting on ODP-90 reverse-phase column, and purification through SP Sepharose HP cation exchange and C2/C18 reverse-phase chromatographies. The antimicrobial was eluted from the C2/C18 column as four individually active fractions, designated A81, B81, C81 and D81. The purification procedure used proved particularly efficient for the bacteriocin in fraction D81, with a yield of 46%, while only 3.8% the bacteriocin in fraction B81 could be collected. MALDI-TOF mass spectrometry of the bacteriocins in fractions B81 and D81 showed respective masses of 4,833.0 and 5,462.2 Da. Amino acid sequencing of the two peptides revealed two class-II bacteriocins whose sequences were similar to those of enterocin A and enterocin B, respectively. Using proper primers, chromosomal fragments of 212 and 216 bp enclosing bacteriocin structural genes were PCR-amplified. Cloning of the amplicons and their sequencing revealed two genes with sequences identical to the structural genes of enterocins A and B, respectively. It was therefore clearly established that E. faecium WHE 81 produces bacteriocins respectively identical to enterocins A and B. Our results, combined with data from previous reports, suggest that the two bacteriocins may be widespread among enterococcal strains and may play an important role in controlling the growth of pathogens and other undesirable bacteria in certain fermented food products.

This short review covers the biotechnological aspects of the production of poly-D-3-hydroxybutyric acid, P(3HB), from H2, O2 and CO2 by autotrophic culture of the hydrogen-oxidizing bacterium, Ralstonia eutropha. Considering the efficiency of utilization of a gas mixture as substrate, a practical fermentation process using R. eutropha for the mass production of P(3HB) from CO2 should be designed on the basis of a recycled-gas, closed-circuit culture system. Also, maintaining the O2 concentration in a gas phase lower than 6.9% (v/v) is essential to prevent the gas mixture from exploding. Our study, using an explosion-proof fermentation bench plant and a two-stage culture system with a newly designed air-lift fermenter, demonstrated that very high P(3HB) yield and productivity could be obtained while the O2 concentration was maintained below 6.9%. However, a study on the continuous production of P(3HB) from CO2 by chemostat culture of R. eutropha revealed that the productivity and content of P(3HB) in the cells was considerably lower than by fed-batch culture. It is deduced that the use of the hydrogen-oxidizing bacterium, Alcaligenes latus, which accumulates P(3HB) even in the exponential growth phase, will be useful for the effective production of P(3HB) from CO2.

Three principal variables, molar ratio of methanol to oil, amount of catalyst, and reaction temperature, affecting the yield of acid-catalyzed production of methyl ester (biodiesel) from crude palm oil were investigated. The biodiesel was then used as an extractant in batch and continuous acetone–butanol–ethanol fermentation, and its fuel properties and that of the biodiesel–ABE product mix extracted from the batch culture analyzed. The optimized variables, 40:1 methanol/oil (mol/mol) with 5% H2S04 (vol/wt) reacted at 95°C for 9 h, gave a maximum ester yield of 97%. Biodiesel preferentially extracted butanol, and enhanced its production in the batch culture from 10 to 12 g l−1. The fuel properties of biodiesel and the biodiesel–ABE mix were comparable to that of No.2 diesel, but their cetane numbers and the boiling points of the 90% fractions were higher. Therefore, they could serve as efficient No. 2 diesel substitutes. The biodiesel–ABE mixture had the highest cetane number.

The effects of several additives on the production of a lantibiotic, nukacin ISK-1, from Staphylococcus warneri ISK-1, in batch fermentation were studied. NaCl, KCl and sorbitol stimulated nukacin ISK-1 production. The addition of 1.4 M NaCl increased nukacin ISK-1 activity 1.5-fold over the control, while cell growth and glucose consumption were inhibited. Nukacin ISK-1 production increased with increasing osmolarity of the medium up to about 3 osmol/kg; however, further increases in osmolarity diminished productivity, irrespective of the kind of additive. Northern blot analysis showed that transcription of the nukacin ISK-1 structural gene (nukA) was activated in the presence of 1.4 M NaC1. These data indicate that the stimulation effect was due to osmotic stress, which acted, at least in part, at the transcriptional level on the nukA gene.

Fermentative production of poly-D-3-hydroxybutyrate [P(3HB)] from a mixture of L-lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l.h.

Nisin Z, a natural nisin variant, is a lantibiotic that contains unusual amino acids. The immobilization technique was applied to improve the productivity of nisin Z, the peptide antibiotic produced by Lactococcus lactis IO-1. Both entrapment and adsorption methods of immobilization were studied with several kinds of materials in repeated fermentation. The long-term application of natural materials was limited because of the instability of the gel. Low growth rate and low productivity were obtained from the entrapment method due to the improper diffusion of nutrients through the materials. Moreover, the method to entrap the cells with photo-crosslinkable resin prepolymers caused damage to the cells. The adsorption method showed better results compared with the entrapment method. The adsorption of the cells on porous chitosan beads, Chitopearl SH-2510, gave nisin Z productivity about 1.7 times greater than the free cells. However, during the repeated cultivation, a decrease in nisin Z activity was observed. The cells adsorbed on photo-crosslinked resin gel beads, ENTG-3800, produced a greater amount of nisin Z and a lower level of lactic acid than the free cells with a good operational stability. The nisin Z production tended to increase with repeated cultivation. Continuous fermentation was introduced to improve the nisin Z productivity. Free cells showed a good productivity at the dilution rate of 0.1 h−1. However, nisin Z production was affected by cell washout at the dilution rate of 0.2 h−1. The cells adsorbed on ENTG-3800 gel beads displayed an improvement in productivity at higher dilution rates.

Two Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ2(Pa) were constructed and introduced into Escherichia coli DH5alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities, that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle.

To investigate the possibility of applying staphylococcal catalase to eliminate H2O2 in lactic acid fermentation, an intracellular catalase from Staphylococcus warneri ISK-1 which exhibited high catalase activity was purified to homogeneity in a six-step purification procedure. The subunit molecular mass of the purified enzyme determined under denaturing conditions was 64,000. The absorption spectrum of the catalase showed a soret band at 406 nm, indicating that the enzyme is a heme protein. Km and Vmax of the catalase for H2O2 were 59 mM and 42,700 U/mg, respectively. The catalase showed a broad optimum pH (5.5–9.5) and stability to organic solvents. We cloned a catalase gene from S. warneri ISK-1 genome. Nucleotide sequence analysis of a 2.2-kb DNA fragment revealed an open reading frame of 1515 bp, called katA, encoding a protein of 504 amino acid residues. The predicted amino acid sequence showed a high homology with other typical catalases. No similarities to bacterial catalase-peroxidase type enzymes were found.

In the last decade, a variety of ribosomally synthesized antimicrobial peptides or bacteriocins produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as producer self protection, membrane-protein interactions, and protein modification and secretion. Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their activities and potential applications. They have first attracted particular attention as listericidal compounds and are now believed to be the next in line if more bacteriocins are to be approved in the future. The present review attempts to provide an insight into general knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances.

A-two stage culture method of hydrogen-oxidizing bacterium, Alcaligenes eutrophus, is used to produce poly-d-3-hydroxybutyrate, P(3HB) from CO2, O2, and H2 without using a very high oxygen transfer rate while maintaining the O2 concentration in gas phase below 6.9 (v/v)% to prevent detonation of the gas mixture. The two-stage method consists of a heterotrophic culture using fructose as carbon source for exponential cell growth and an autotrophic culture for P(3HB) accumulation. We investigated the use of acetic acid as a cheaper carbon source than fructose for the heterotrophic culture in the two-stage method. However, the acetate concentration in the culture system must be maintained at 1.0 g · dm−3 since its inhibitory effect on the cell growth is very strong. Then, high cell density cultivation of A. eutrophus was investigated by pH-stat continuous feeding of acetic acid to control acetate concentration. As a result, acetate concentration was automatically maintained around 1.0 g · dm−3 by using a feed with a composition in CH3COOH/CH3COONH4/KH2PO4 molar ratio of 5:1:0.084. Cell concentration increased to 48.6 g · dm−3 after 21 h of cultivation. The cell mass grown in the fed-batch culture on acetic acid was useful for P(3HB) production from CO2 in the subsequent autotrophic culture stage. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 625–631, 1999.

The possibility of employing methylated crude palm oil (CPOE) as an extraction solvent to reduce end-product inhibition and to enhance solvent productivity in acetone-butanol-ethanol (ABE) fermentation was evaluated using oleyl alcohol as the standard butanol extractant. Fermentation was carried out at an initial glucose concentration of 90 g/l. CPOE did not inhibit the growth of the fermentative organism. Without solvent extraction, butanol production ceased after 30 h at a concentration of 15.4 g/l limiting cell growth to 3.98 g/l and glucose consumption to 62%. Applying CPOE as the extraction solvent, about 47% of the total butanol produced was extracted, glucose consumption was increased to 83% and relatively high glucose consumption rates and solvent productivities were obtained. Butanol production increased to 20.9 g/l; total ABE solvents and yield also increased from 21.2 g/l and 38% (in conventional fermentation) to 29.8 g/l and 40.4%, respectively.

The utility of the juice of rotten or discarded pineapples and the waste material of the production of pineapple juice as low-cost substrates for ethanol production by Zymomonas mobilis was investigated. Z. mobilis ATCC 10988 produced 59.0g·l −1 ethanol in undiluted pineapple juice without nutritional supplementation and without the regulation of the pH while 42.5g·l −1 ethanol was obtained using a125g·l −1 sucrose medium supplemented with 10g·l −1 yeast extract and mineral salts. Ethanol fermentation using unhydrolyzed and enzymatically hydrolyzed pineapple waste material was also investigated under various culture conditions. When a 15% (v/v) dilution of unhydrolyzed waste material without nutritional supplementation was used, more than 3.5g·l −1 ethanol was produced. When the media containing 15, 30, and 40% (v/v) of the hydrolyzate consisting of a 60% (v/v) suspension of pineapple waste material were used, final concentrations of ethanol were 5.0g·l −1 , 7.6g·l −1 , and 9.3g·l −1 , respectively. These results suggest that pineapple juice and the waste material can be useful low-cost substrates for ethanol production by Z. mobilis without supplementation with expensive organic nitrogen complexes such as yeast extract and without the regulation of the pH during cultivation, leading to the reduction in the production costs.

Natural rubber serum powder, rich in crude protein and carbohydrates, had a strong growth-stimulating activity for Bifidobacterium bifidum JCM 1254, which was unable to grow in a fully synthetic medium, B12 assay medium. Natural rubber serum powder was fractionated by ultrafiltration (molecular weight cutoff 1000). The active ultrafiltrate was further concentrated and desalted with an adsorptive microconcentrator, which adsorbs virtually all amino acids and peptides. Through this purification step, it was found that the adsorbed fraction obtained did not stimulate growth independently but acted complementarily with a small amount of ammonium sulfate. The adsorbed fraction was subsequently analyzed on reversed-phase high pressure liquid chromatography, and the activities of the eluates were measured on B12 assay medium with ammonium sulfate. Consequently, it was proved that several peptidic ingredients in the adsorbed fraction increased the growth of B. bifidum.

Conversion of xylose to l -lactate was carried out by Lactococcus lactis IO-1 using an electrodialysis bioprocess (ED-BP). At 50 g l −1 xylose, the ED-BP was already complete in half the time (32 h) taken by the control culture without electrodialysis (>60 h). At 80 g l −1 xylose, the control culture was unable to consume >50 g l −1 xylose, whereas the ED-BP consumed 75 g l −1 xylose in 45 h. Thus, the simultaneous removal of lactate and acetate by ED-BP was associated with high-speed l -lactate production, increased xylose consumption and an increased l -lactate production.

Lactococcus lactis IO-1 isolated in our laboratory produces a peptide antibiotic, a natural nisin variant, nisin Z. Nisin Z was heat stable at acidic pH and showed a bactericidal mode of action against an indicator strain, Bacillus subtilis C1. The effect of proteolytic enzyme treatment on the activity and the antimicrobial spectrum were tested. These results indicated nearly the same characteristics as those of nisin A. In this study, it was found that two nisin variants (nisin Z and nisin A) were practically inactivated by proteinase K and actinase E. Interestingly, nisin-producing strains, L. lactis IO-1 and L. lactis NCDO 497 (a nisin A producer) were sensitive to a high concentration of nisin variants including their own products. The growth of a Gram-negative bacterium, Escherichia coli, was also inhibited by a high concentration of nisin, although nisin producers and Gram-negative bacteria are generally resistant to nisin variants.

Culture systems involving the use of one or two fermenters in series, with cell recycle employing hollow fiber filter modules to achieve high cell densities, were developed. Dilution rates of about 0.6 h -1 and ethanol productivity values of about 25 g/l h -1 , with residual glucose at 0.9 g/l, could be achieved using the system employing one fermenter.

The growth of a newly isolated strain of Pediococcus sp., designated ISK-1, was very slow and the concentration of cells in the medium remained low. Fermentation with an initial 30 g/liter glucose required about 60 h. To stimulate fermentation, we attempted to optimize the medium by flask culture and jar fermentation tests. Mevalonic acid and mieki (soy bean hydrolyzate) stimulated fermentation and increased the rate of formation of DL-lactate. Kinetic analysis of the fermentation showed that mevalonic acid markedly increased the specific glucose consumption rate and the specific lactate production rate. Mieki and mevalonic acid had a synergistic effect, but the effect of mevalonic acid was different from that of mieki.