Isolation and characterization of a new <Emphasis Type="Italic">Bacillus</Emphasis> sp. 50-3 with highly alkaline keratinase activity from <Emphasis Type="Italic">Calotes versicolor</Emphasis> faeces

A new native feather-degrading bacterium has been isolated from the faeces of the agamid lizard Calotes versicolor, collected from the Beijing Zoo in China. The isolate, which has been identified as Bacillus sp. 50-3 based on morphological and biochemical and 16S rDNA tests, was shown to degrade native feather completely at 37°C and pH 7.0 within 36 h when using chicken feathers as the sole carbon and nitrogen source. Bacillus sp. 50-3 presented optimum growth at 37°C and pH 7.0 in feather meal medium. Under these conditions, the maximum keratinase activity (680 ± 25 U/ml) was also achieved. The keratinase of Bacillus sp. 50-3 was active over a broad range of pH values and temperatures toward azokeratin, and presented an optimum pH and temperature of 10.0 and 60°C, respectively. Furthermore, it was relatively heat-and alkali-stable. Inhibitor studies showed that it seemed to belong to the serine-metalloprotease type. Therefore, the enzyme from Bacillus sp. 50-3 is a novel, high alkaline keratinase, suggesting its potential use in biotechnological processes.     

Characterization of two bacteriocins produced by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages

Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages, produce bacteriocins antagonistic towards closely related species and pathogens, such as Listeria monocytogenes. The bacteriocins were inactivated by proteolytic enzymes and lipase but not by catalase and lysozyme. They were also heat stable, retaining activity after heating at 100 °C for 60 min. The bacteriocins were stable at pH values ranging from 2.0 to 8.0. Bacteriocin production was observed at low temperatures (10 and 4 °C) and in meat juice. The maximum bacteriocin activity was observed at the end of the exponential growth phase. The bacteriocins were produced in media with initial pH values ranging from 5.0 to 7.5, but not in media with a pH lower than 5.0 (weak bacteriocin activity of the antibacterial compound produced by Ln. mesenteroides L124 was observed at pH 4.5). Both bacteriocins exhibited strong bactericidal activity following cell/bacteriocin contact.     

Statistical optimization of physical parameters for enhanced bacteriocin production by L. casei

Antimicrobial proteinaceous compounds such as bacteriocins produced from Lactobacillus sp. are widely known. They have potential antimicrobial activities towards closely related bacteria and several pathogens associated with food spoilage and hence can be a potential food bio-preservative agent. Bacteriocin production requires optimized process, complex media and well-controlled physical conditions including pH and temperature. A probiotic strain of L. casei LA-1 isolated from mango pickle was used in the present study. The influence of physical parameters viz. temperature (15 ?? 45°C), pH (4.0 ?? 7.0), incubation time (up to 48 h) and inoculum size (0.7 ?? 2.0 O.D) on bacteriocin production was analyzed. The effect of all the parameters was first investigated using the one-factor-at-a-time method (OFAT) to see the significance of these parameters on bacteriocin production and then further optimized by response surface methodology (RSM). Following OFAT analysis, all factors were found to have a significant effect on bacteriocin production. Bacteriocin production of 2,844 AU/mL was obtained at temperature 37°C, pH 6.7 and inoculum size 1.8 O.D at an incubation time of 20 h and it was produced during the stationary phase of growth. Statistical analysis showed that three variables-pH, temperature and incubation time have significant effects on bacteriocin production. RSM proved to be a powerful tool in the optimization of bacteriocin production by L. casei LA-1 with a two-fold increase, giving a production of 4652.15 AU/mL at pH 7.19, temperature 33.3°C and incubation time of 22.2 h.     

Bacillus fermentation of soybeans

Kinema fermentations of Indian and Canadian No. 1 soybeans by Bacillus sp. DK-W1 and by mixed cultures of Bacillus sp. DK-W1 and Enterococcus faecium DK-C1 were essentially identical. The viable cell count of Bacillus increased from an initial 10⁵ to 10¹⁰ c.f.u./g wet wt after 48 h incubation at 37°C. The pH of the fermentation dropped from an initial 6.9 to about 6.4 after 8 h and then rose to 8.6 after 32 h, with a coincident increase in proteolytic activity and ammonia concentration. The fermentations containing E. faecium and Bacillus exhibited a greater initial pH decline and a slightly retarded subseqent increase in pH compared with fermentations with Bacillus only. The presence of E. faecium had no detectable effects on growth of the Bacillus, proteolytic activity, ammonia production or the final pH of the fermentations.P.K. Sarkar was and P.E. Cook and J.D. Owens are with the Food Microbial Interactions Laboratory, Department of Food Science and Technology, University of Reading, Reading RG6 2AP, UK; P.K. Sarkar is now with the Microbiology Laboratory, Centre for Life Sciences, University of North Bengal, Siliguri 734430, India.     

Addendum to Issue 1 - ENZITEC 2012 Cheese whey and passion fruit rind flour as substrates for protease production by Bacillus sp. SMIA-2 strain isolated from Brazilian soil

Abstract

Application of wastes from the food processing industry as carbon sources in enzyme production processes reduces the cost of production, and also helps in solving problems of their disposal. In this work, we demonstrated that sweet cheese whey, in combination with passion fruit rind flour, can be successfully used for the production of protease by Bacillus sp. SMIA-2, opening perspectives for the use of these agricultural byproducts as novel and cost-effective culture media for the production of protease. The maximum production of the enzyme was observed in a sweet cheese whey-based culture medium preparation (0.5%, w/v) containing 0.25% (w/v) passion fruit rind flour and supplemented with different metal salts at an initial pH of 7.5–8.0, incubated at 50°C for 48 h. Studies on enzymatic characterization revealed that crude protease showed maximum activity at pH 9.0 and 70°C. These characteristics presented by the protease produced by Bacillus sp. SMIA-2 could be very useful when thinking about biotechnological applications.     

Influence of carbon and nitrogen sources on antifungal metabolite production by bacterium associated with entomopathogenic nematode against Penicillium expansum

A specific symbiotic Bacillus sp. isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce large number of bioactive compounds. The present study was conducted to determine the effect of carbon and nitrogen sources for the production of antimicrobial substances by Bacillus sp. The yield of the crude antimicrobial substances and antimicrobial activity against the test micro-organism also differed significantly when carbon and nitrogen sources in the fermentation media were changed. The antifungal activity was significantly high in yeast extract plus fructose (46.5?±?2.12?mm) followed by yeast extract plus maltose, beef extract plus fructose and meat infusion plus glucose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention time indicating that they produced different compounds. When the carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced. The results indicate that carbon source in the fermentation media plays a vital role in the production of antifungal substances. It is concluded that yeast extract and fructose as nitrogen and carbon sources produced maximum activity, which can effectively control the blue mould caused by Penicillium expansum in apples and pears.     

Bacillus sp. mutant for improved biodegradation of Congo red: Random mutagenesis approach

This study presents the improved biodegradation of Congo red, a toxic azo dye, using mutant Bacillus sp. obtained by random mutagenesis of wild Bacillus sp. using UV and ethidium bromide. The mutants obtained were screened based on their decolorization performance and best mutants were selected for further studies. Better decolorization was observed in the initial Congo red concentration range 100–1000 mg/l for wild species whereas mutant strain was found to offer better decolorization up to 3000 mg/l. Mutant strain offered 12–30% reduction in time required for the complete decolorization by wild strain. The optimum pH and temperature were found to be 7.0 and 37 °C, respectively. Two efficient strains such as Bacillus sp. ACT 1 and Bacillus sp. ACT 2 were isolated from the various mutants obtained. Bacillus sp. ACT 2 showed improved enzymatic production and Bacillus sp. ACT 1 showed improved growth compared to wild strain. The enzyme responsible for the degradation was found to be azoreductase by SDS–PAGE and about 53% increased production of enzyme was achieved with mutant species. The experimental data were modeled using growth and substrate inhibition models.     

Isolation and characterisation of phosphate solubilising microorganisms from the cold desert habitat of <Emphasis Type="Italic">Salix alba Linn.</Emphasis> in trans Himalayan region of Himachal Pradesh

Phosphate solubilising microorganisms (PSM) (bacteria and fungi) associated with Salix alba Linn. from Lahaul and Spiti valleys of Himachal Pradesh were isolated on Pikovskaya (PVK), modified Pikovskaya (MPVK) and National Botanical Research Institute agar (NBRIP) media by spread plating. The viable colony count of P-solubilising bacteria (PSB) and fungi (PSF) was higher in rhizosphere than that of non-rhizosphere. The frequency of PSM was highest on MPVK followed by NBRIP and PVK agar. The maximum proportion of PSM out of total bacterial and fungal count was found in upper Keylong while the least in Rong Tong. The PSB frequently were Gram-positive, endosporeforming, motile rods and belonged to Bacillus sp. The PSF mainly belonged to Penicillium sp., Aspergillus fumigatus, A. niger, A. spp. and non-sporulating sterile. Amongst the isolates with high efficiency for tricalcium phosphate (TCP) solubilisation, seven bacterial and seven fungal isolates dissolved higher amount of P from North Carolina rock phosphate (NCRP) than Mussoorie rock phosphate (MRP) and Udaipur rock phosphate (URP). However, the organisms solubilised higher-P in NBRIP broth than PVK broth. SBC5 (Bacillus sp.) and SBC7 (Bacillus sp.) bacterial isolates exhibited maximun P solubilisation (40 and 33 μg ml⁻¹ respectively) whereas FC28 (Penicillium sp.) isolate (52.3 μg ml⁻¹) amongst fungi while solubilising URP. The amount of P solubilised was positively correlated with the decrease in pH of medium. SBC5 (Bacillus sp.), SBC7 (Bacillus sp.) and SBC4 (Micrococcus) decreased the pH of medium from 6.8 to 6.08 while FC28 (Penicillium sp.) and FC39 (Penicillium sp.) isolates of fungi recorded maximum decrease in pH of medium from 6.8 to 5.96 in NBRIP broth.     

Resident bacteria, nitric oxide emission and particle size modulate the effect of Brassica napus seed meal on disease incited by Rhizoctonia solani and Pythium spp

Amendment of orchard soil with low-glucosinolate Brassica napus (rape) seed meal (RSM) suppresses infection of apple roots by Rhizoctonia solani but increases incidence of Pythium spp. infection. Following incorporation of Brassica sp. seed meals, soils were monitored for changes in populations of selected saprophytic and plant pathogenic microorganisms. When conducted in pasteurized soil, which possessed high numbers of Bacillus spp. and lower than detectable numbers of Streptomyces spp., RSM amendment did not provide control of R. solani. Populations of streptomycetes in RSM-amended soil increased to stable levels >20-fold higher than in non-amended soil. Disease suppressiveness was restored to pasteurized RSM-amended soil by adding any of several Streptomyces strains. Maximal rates of nitrification in orchard soil, determined by nitric oxide emission, were observed within two weeks following RSM amendment and inhibition of nitrification via application of nitrapyrin abolished the capacity of RSM to suppress R. solani infection of apple roots when seedlings were planted one day after soil amendment. Apple seedling mortality and Pythium spp. root infection were highest for seedlings planted immediately following incorporation of B. napus cv. Athena RSM, particularly when meal was added in a flake rather than powder form. Lower infection frequencies were observed for seedlings planted four weeks after RSM incorporation, even for soil in which densities of culturable Pythium spp. had not declined. Our results demonstrate that suppression of Rhizoctonia root rot in response to RSM amendment requires the activity of the resident soil microbiota and that initial disease control is associated with the generation of nitric oxide through the process of nitrification.     

Antifungal activity of crude extract produced by Bacillus sp. associated with entomopathogenic nematode from media formulated by six nitrogen sources with fructose against Penicillium expansum

A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different nitrogen sources in combination with fructose on the production of antifungal crude extract by Bacillus sp. against Penicillium expansum. The yield of crude extract and antifungal activity against the test fungi differed significantly when the nitrogen sources in the fermentation media were changed. The highest yield was recorded for beef extract plus fructose (921?mg/L). The antifungal activity was higher in yeast extract plus fructose [P. expansum (46.5?±?2.12?mm)], followed by beef extract. High performance liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation medium, the antimicrobial production was substantially reduced almost eight times. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Yeast extract and fructose as nitrogen and carbon sources in the fermentation medium produced maximum antimicrobial activity.     

Optimization of antifungal lipopeptide production from Bacillus sp. BH072 by response surface methodology

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH₄Cl, urea, and ammonium citrate); and metal ions (Zn²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ca²⁺, and K⁺). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg²⁺ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg²⁺. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.     

Impact of beef extract and six carbon source on antifungal metabolites production by bacterium associated with entomopathogenic nematode against Fusarium oxysporum

A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp., was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different carbon sources in combination with beef extract on the production of antifungal substances by Bacillus sp. The yield of crude antimicrobial substances and antimicrobial activity against the test microorganism also differed significantly when the carbon sources in the fermentation media were changed. The highest yield was recorded for fructose plus beef extract (956?mg/l). The antifungal activity was significantly high in beef extract plus maltose (21?±?1.5?mm) followed by beef extract plus glucose and beef extract plus fructose. Antifungal activity was significantly reduced in beef extract plus lactose and sucrose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation media, the antifungal production was substantially reduced. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Beef extract and maltose as nitrogen and carbon sources in the fermentation medium produced maximum antifungal activity. It is concluded that Beef extract and maltose as nitrogen and carbon sources produced maximum activity which can effectively control the Fusarium oxysporum which causes vascular fusarium wilt in tomato, tobacco, legumes, cucurbits, sweet potatoes, banana, etc.     

Optimization of extracellular alkaline protease production from species of <Emphasis Type="Italic">Bacillus</Emphasis>

Thirty-five strains capable of secreting extracellular alkaline proteases were isolated from the soil and waste water near the milk processing plant, slaughterhouse. Strain APP1 with the highest-yield alkaline proteases was identified as Bacillus sp. The cultural conditions were optimized for maximum enzyme production. When the initial pH of the medium was 9.0, the culture maintained maximum proteolytic activity for 2,560 U ml⁻¹ at 50°C for 48 h under the optimized conditions containing (g⁻¹): soyabean meal, 15; wheat flour, 30; K₂HPO₄, 4; Na₂HPO₄, 1; MgSO₄·7H₂O, 0.1; Na₂CO₃, 6. The alkaline protease showed extreme stability toward SDS and oxidizing agents, which retained its activity above 73 and 110% on treatment for 72 h with 5% SDS and 5% H₂O₂, respectively.     

Inhibitory activity against the fish pathogen Lactococcus garvieae produced by Lactococcus lactis TW34, a lactic acid bacterium isolated from the intestinal tract of a Patagonian fish

After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the antimicrobial production was as low as 15°C. Both acidification and hydrogen peroxide production were ruled out as the source of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121°C for 15 min) and active between pH 3 and 11. It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C. Our results suggest that the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish.     

Influence of pH and temperature on the expression of sboA and ituD genes in Bacillus sp. P11

Temperature and pH are key factors influencing the production of antimicrobial peptides. In this work, qRT-PCR methodology was used to demonstrate the effect of these two variables on sboA (subtilosin A) and ituD (iturin A) expression in Bacillus sp. P11, an isolate from aquatic environment of the Amazon. Bacillus sp. P11 was incubated in BHI broth for 36 h at 30, 37 and 42 °C, and the pH values were 6.0, 7.4 and 8.0. The production of subtilosin A and iturin A was confirmed by mass spectrometry. The sboA expression increased 200-fold when the initial pH was 8.0. In contrast, ituD expression was maximum at pH 6.0. Increased temperature (42 °C) was adverse for both genes, but ituD expression increased at 37 °C. Expression of sboA and ituD was strongly affected by pH and temperature and qRT-PCR proved to be a powerful tool to investigate the potential of Bacillus strains to produce subtilosin A and iturin A.     

Characterization of a broad range antibacterial substance from a new <Emphasis Type="Italic">Bacillus</Emphasis> species isolated from Amazon basin

A Bacillus sp. strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. The phylogenetic analysis indicated that this strain has low sequence similarity with most Bacillus spp., suggesting a new species was isolated. The antimicrobial activity was detected starting at the exponential growth phase, and maximum activity was observed at stationary phase. The substance was inhibitory to a broad range of indicator strains, incluing pathogenic and food spoilage bacteria such as Listeria monocytogenes, B. cereus, Aeromonas hydrophila, Erwinia carotovora, Pasteurella haemolytica, Salmonella Gallinarum, among other. The antibacterial substance was stable over a wide pH range, but it was sensitive to pronase E and lipase. The antibacterial substance was bactericidal and bacteriolytic to L. monocytogenes and B. cereus at 160 AU ml⁻¹. The identification of a broad range bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage microorganisms.     

Isolation and characterization of a marine algicidal bacterium against the harmful raphidophyceae <Emphasis Type="Italic">Chattonella marina</Emphasis>

A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of ULjin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal activity by different culture filtrate volumes, the 10% (100 μl/ml) concentration showed the biggest change in algicidal activity; there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5. Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina.     

Influence of media and temperature on bacteriocin production by<Emphasis Type="Italic"> Bacillus cereus</Emphasis> 8A during batch cultivation

Cerein 8A is a bacteriocin produced by the soil bacterium Bacillus cereus 8A, isolated from native woodlands of Brazil. The influence of temperature and media on the growth of B. cereus 8A and the production of this bacteriocin was studied during batch cultivation. Maximum activity was detected by cultivation in brain/heart infusion broth, reaching 3200 activity units ml^–1. Bacteriocin was also produced in peptone, MRS, Mueller–Hinton and nutrient broth, while no activity was observed during cultivation in thioglycollate or tryptic soy broth. Temperature had a strong influence on bacteriocin production, which was higher at 30 °C than at 25 °C. An important decrease in bacteriocin activity was observed at 37 °C. The relationship between growth and specific production rates, as a function of the temperature, showed different kinetics of production and there were several peaks in the specific production rates during growth. Bacteriocin was produced at the stationary phase, indicating it is synthesized as a secondary metabolite.     

Growth conditions required for bacteriocin production by strains of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The influence of growth parameters on the production of bacteriocins (aureocins) by five strains of Staphylococcus aureus was investigated. These aureocins have a broad spectrum of activity and can inhibit important human and animal pathogens. All strains produced large inhibition zones upon the indicator strain when they were grown in rich media such as brain-heart infusion (BHI), N2GT and 2 × YT. Bacteriocin production was not influenced by the initial pH of the medium (6.0–8.0). At lower temperature (28 °C), there was a marked reduction in bacteriocin production. Incubation of the producers under anaerobiosis affected profoundly the production of two related bacteriocins, aureocins MB92 and 146L, and slightly increased the production of aureocins A53 and 215FN. Production of aureocins MB92 and 215FN was apparently abolished in media containing 2.5 g and 3.5 g Nacl/100 ml, respectively. Although production of the remaining aureocins was observed in all NaCl concentrations tested (0.5–7.5 g/100 ml), the larger inhibition zones were detected in media containing up to either 1.5 g (for aureocins A70 and 146L) or 2.5 g NaCl/100 ml (for aureocin A53). Aureocin 215FN could not be detected in the culture supernatant. For the remaining aureocins tested, the highest levels of bacteriocin production occurred in either the late-log or early stationary growth phase of cultures grown in BHI medium at 37 °C. Changes in environmental conditions can, therefore, have detrimental effects on the production of active aureocins. Such factors are relevant when considering the potential biotechnological applications of these substances and when testing new S. aureus isolates for bacteriocin production.     

Carnocin KZ213 produced by<Emphasis Type="Italic"> Carnobacterium piscicola</Emphasis> 213 is adsorbed onto cells during growth. Its biosynthesis is regulated by temperature,pH and medium composition

Carnocin KZ213 is an antilisterial bacteriocin produced by Carnobacterium piscicola 213. The effects of pH and temperature were studied during batch fermentation in MRS* medium (modified MRS without ammonium citrate or sodium acetate). The optimal pH for growth is between 6 and 7. The maximum bacteriocin productivity in the supernatant occurs at pH 7. Operating at controlled pH increases the volumetric activity of the free bacteriocin by 8- to 16-fold, compared with uncontrolled pH. No bacteriocin production is observed below pH 6.5. Temperature has a dramatic effect on carnocin KZ213 production. Growth is optimal at 25 °C and 30 °C, although no bacteriocin production is detected at 30 °C. Also, bacteriocin production is observed at 25 °C in MRS*, but not in complex APT broth, where growth is optimal. The presence of glucose as a carbon and/or energy source is important for carnocin KZ213 synthesis. Hence, bacteriocin synthesis is regulated by temperature, carbon source and medium composition. Quantification studies of bacteriocin adsorbed onto producer cells show that the majority of the carnocin KZ213 secreted is adsorbed onto the producer cells during growth. Only 15% of the total bacteriocin produced is detected in the cell-free supernatant at the end of growth.