Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999     

Influence of Extracellular Polymeric Substances on the Adsorption of Cadmium onto Three Bacterial Species

In this study, we measured the effect of EPS on Cd and proton adsorption behaviors by measuring the extent of adsorption onto biomass with and without the EPS removed via a cation exchange resin. We conducted both Cd adsorption experiments and potentiometric titrations of biomass using three common bacterial species: one Gram-positive (Bacillus subtilis) and two Gram-negative (Shewanella oneidensis, Pseudomonas putida) species. The Cd adsorption experiments were conducted as a function of metal loading in order to probe whether environmentally-low metal loadings lead to different adsorption mechanisms and roles for EPS than the higher metal loadings of most previous adsorption studies. We suspended each biomass sample in a solution of dissolved Cd in 0.01?M NaClO₄ at metal loadings of 1, 2, 5, and 74?μmol/g. Surface complexation modeling (SCM) was used to determine stability constants for the important Cd-bacteria complexes, and the effect of metal loading on the resulting calculated stability constant values was determined.

In general, the measured bulk Cd adsorption behavior is unaffected by EPS removal. However, our potentiometric titration results suggest that EPS removal does alter the distribution of site types, but not the mass-normalized total site concentration within the biomass. SCM suggests that high affinity sulfhydryl sites control Cd binding under low metal loading conditions for B. subtilis and P. putida, and that sulfhydryl sites are present both on the cells and within the EPS for these species. Conversely, the SCM results suggest that Cd-sulfhydryl binding is un-important on the EPS of S. oneidensis.     

Experimental Measurements of the Adsorption of Bacillus subtilis and Pseudomonas mendocina Onto Fe-Oxyhydroxide-Coated and Uncoated Quartz Grains

In this study, we compared the adsorption of the gram-positive bacterium Bacillus subtilis with adsorption of the gram-negative bacterium Pseudomonas mendocina onto Fe-oxyhydroxide-coated and uncoated quartz grains as a function of pH and bacteria: mineral mass ratio. We studied metabolically-inactive cells in order to focus on the initial bacterial attachment mechanisms. The data show that the presence of Fe-oxyhydroxide-coatings on quartz surfaces significantly enhances the adsorption of bacteria and that in general the extent of adsorption decreases with increasing pH and with decreasing bacteria: mineral mass ratio. B. subtilisadsorbs to a greater extent than does P. mendocina onto the surface of the Fe-coated quartz. The adsorption behavior appears to be controlled by the overall surface charge of both bacterial and mineral surfaces. We model the adsorption data using a semi-empirical chemical equilibrium model that accounts for the site speciation of the adsorbing surfaces. Models describing bacterial adsorption to Fe-oxyhydroxide-coated quartz can account for changes in pH and bacteria: mineral mass ratio using one set of equilibrium constants.     

Bacillus subtilis secretes a foreign protein by the signal sequence of Bacillus amyloliquefaciens neutral protease

Summary We constructed a secretion plasmid in which a truncated penicillinase gene of Bacillus licheniformis was introduced at the end of the signal peptide coding region of a Bacillus amyloliquefaciens neutral protease gene. A Bacillus subtilis recombinant secreted about 140 mg/liter of the penicillinase into the medium. Analysis of the purified product revealed that it was a mixture of two penicillinases containing one or two additional amino acids at the NH₂-terminus of B. licheniformis exo-small penicillinase.     

Immunological comparison of bacterial α-amylases and multiple forms of Baci1lus licheniformis enzyme

A purified preparation of Bacillus licheniformis α-amylase was immunologeeally and electrophoretically compared with commercial crystalline α-amylase of Bacillus subtilis. The former enzyme reacted completely with rabbit antiserum to the same enzyme showing a single precipitin band, and moved toward the cathode in immuno-electrophoresis on agarose at pH 9.6. On the contrary, crystalline α-amylase of Bacillus subtilis migrated to the anode in immunoelectrophoresis at pH 8.6, though it weakly cross-reacted with the antiserum, suggesting that amylases of Bacillus licheniformis and Bacillus subtilis are not identical. In addition, the neutralization test of amylase activity showed that α-amylase of Bacillus licheniformis was much more susceptible to inhibition by the serum than was Bacillus subtilis α-amylase. Each of four species of Bacillus licheniformis α-amylase extracted from the sliced discs after disc electrophoresis on polyacrylamide gel was distinct from the others by showing individual migratory rate, but they were antigenically similar to each other and to the parent enzyme.     

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species

Background

Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.

Results

We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.

Conclusions

Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.     

Potato peel as a solid state substrate for thermostable α-amylase production by thermophilic Bacillus isolates

Summary Potato peel was found to be a superior substrate for solid state fermentation, compared to wheat bran, for the production of α-amylase by two thermophilic isolates of Bacillus licheniformis and Bacillus subtilis. Under optimal conditions, B. licheniformis produced 270 units/ml and 175 units/ml of α-amylase on potato peel and wheat bran, respectively, while the corresponding values for B. subtilis were 600 units/ml and 265 units/ml. The enzyme from B.␣licheniformis was optimally active at 90 °C and pH 9.0, while that from B. subtilis at 60 °C and pH 7.0. The nature of the experimental data permitted excellent polynomial fits, on the basis of which, two master equations, corresponding to the isolated strains, were derived for estimation of enzyme activity for any set of values of temperature, particle size, moisture, and incubation time within the indicated ranges.     

Cloning and overexpression of <Emphasis Type="Italic">aprE3-17</Emphasis> encoding the major fibrinolytic protease of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> CH 3-17

Bacillus licheniformis (B. licheniformis) CH3-17, an isolate from cheonggukjang, a traditional Korean fermented soyfood, secretes several fibrinolytic enzymes into the culture medium, showing strong fibrinolytic activity. A gene homologous to aprE of Bacillus subtilis (B. subtilis), aprE3-17, was cloned by PCR. DNA sequencing showed that aprE3-17 encodes a prepro-type serine protease consisting of 382 amino acids. The mature enzyme was 27 kDa in size. The aprE3-17 gene was overexpressed in B. subtilis WB600 using pHY300PLK, an Escherichia coli (E. coli)-Bacillus shuttle vector, and the 27 kDa enzyme was purified from the culture supernatant. The optimum pH for activity was 6.0. Purified enzyme quickly degraded the Aα and Bβ chains of fibrinogen but could not degrade the γ-chain.     

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.     

Nucleotide Sequence and Features of the Bacillus licheniformis gnt Operon

Bacillus licheniformis was able to utilize gluconate as thesole carbon source as efficiently as Bacillus subtilis did.Southern analysis indicated that B. licheniformis likely possessesonly one gnt determinant. The nucleotide sequence (6278 bp)of the B. licheniformis DNA containing the gnt operon was determined,revealing the five complete open reading frames (ORF; genes).The putative product of the first gene, oug, did not show anysignificant homology to known proteins, but those of the secondto fifth genes exhibited striking homology to the gntRKPZ genesof B. subtilis, respectively, indicating that they are the correspondinggnt genes of B. licheniformis. Not only is the organizationof the gnt genes of these two Bacilli highly conserved, butso are the cis regulatory elements of their gnt operon. Sequenceanalysis of the upstream regions of these two gnt operons impliedthat a chromosome rearrangement in B. subtilis might have occurredimmediately upstream of the gnt operon during evolution, causingit to diverge from a common ancestor into B. licheniformis andB. subtilis.     

Kinetic and Equilibrium Studies of Cr(VI) Biosorption by Dead <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Biomass

Many studies have been carried out on the biosorption capacity of different kinds of biomass. However, reports on the kinetic and equilibrium study of the biosorption process are limited. In our experiments, the removal of Cr(VI) from aqueous solution was investigated in a batch system by sorption on the dead cells of Bacillus licheniformis isolated from metal-polluted soils. Equilibrium and kinetic experiments were performed at various initial metal concentrations, pH, contact time, and temperatures. The biomass exhibited the highest Cr(VI) uptake capacity at 50°C, pH 2.5 and with the initial Cr(VI) concentration of 300 mg/g. The Langmuir and Freundlich models were considered to identify the isotherm that could better describe the equilibrium adsorption of Cr(VI) onto biomass. The Langmuir model fitted our experimental data better than the Freundlich model. The suitability of the pseudo first-order and pseudo second-order kinetic models for the sorption of Cr(VI) onto Bacillus licheniformis was also discussed. It is better to apply the pseudo second-kinetic model to describe the sorption system.     

Screening of waste biomass from Saccharomyces cerevisiae,Aspergillus oryzae and Bacillus lentus fermentations for removal of Cu,Zn and Cd by biosorption

Three by-products of fermentations containing Bacillus lentus, Aspergillus oryzae or Saccharomyces cerevisiae biomass were tested for the capacity to absorb Cu, Cd and Zn. The composition of the three biomasses was first determined and showed high contents of ashes in both B. lentus and A. oryzae biomass and high amounts of lipids in the bacterial biomass. Metal ion binding experiments were performed by contact of 0.1 g of biomass (protonated for all the metal tests and not protonated only for the Cd test) with 50 ml of solutions containing each of the metals in the concentration range from 10 to 500 mg/ml, at pH 4.5, 3.5 and 2.5. The final metal ion concentrations were determined using a plasma absorption spectrometer, and the metal removal levels for isotherm plots were determined using the Langmuir model. The results showed that B. lentus protonated biomass had the best sorption capacity for Cu and Cd, followed by protonated A. oryzae and S. cerevisiae biomass. The sorption of Zn was low for all tested biomasses, as also was the binding of all metals at acidic pH (2.5 and 3.5). A significant increase in Cd sorption was obtained using non-protonated biomass from B. lentus and A. oryzae.     

Regulation of Polyglutamic Acid Synthesis by Glutamate in Bacillus licheniformis and Bacillus subtilis

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular γ-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.     

The Effects of Bacterial Surface Adsorption and Exudates on HgO Precipitation

The effects of nonmetabolic bacterial cell wall adsorption and the presence of bacterial exudates on the precipitation of mineral phases from solution is not well constrained experimentally. In this study, we measured the extent of Hg(II) removal from solution, in the presence and absence of nonmetabolizing cells of Bacillus subtilis in both Cl-free and Cl-bearing systems with Hg concentrations ranging from undersaturation to supersaturation with respect to montroydite [HgO_(s)]. Total Hg molalities ranged from 10^?5.00 to 10^?2.00 M at pH 4.50 and 7.00; the ionic strength of the experiments was kept constant using 0.01 M NaClO₄, and the wet mass of bacteria was held constant at 5 g/L for each biotic experiment.

The biotic systems exhibited enhanced Hg(II) removal from solution relative to the abiotic controls in undersaturated conditions. However, thermodynamic modeling of the experimental systems strongly suggests that all of this Hg removal can be ascribed to Hg adsorption onto cell envelope functional groups. There was no evidence for enhanced Hg removal due to precipitation in bulk solutions that were undersaturated with respect to the solid phase. Under the highest total Hg concentrations studied in both the Cl-free and Cl-bearing systems, bacteria inhibit precipitation, maintaining high concentrations of Hg in solution. Cell-free, exudate-bearing control experiments suggest that aqueous complexation between Hg and the bacterially-produced exudates accounts for at least some of the precipitation inhibition. However, a comparison of total available binding sites on the exudates with the concentration of Hg in solution suggests that aqueous complexation alone can not account for the observed elevated final aqueous Hg concentrations in solution, and that the exudates likely exert a kinetic inhibition on the precipitation reaction as well.     

Reconstitution of active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis.

Active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis can be reconstituted in vitro from dissociated RNA and proteins. The reconstituted 50 S sub-units are indistinguishable from native 50 S subunits in sedimentation on sucrose gradients and in protein composition. The procedure used is similar to that developed for reconstitution of Bacillus stearothermophilus 50 S subunits, though the optimal conditions are somewhat different. Hybrid ribosomes can be reconstituted with 23 S RNA and proteins from different sources (B. stearothermophilus and B. licheniformis or B. subtilis). The thermal stability of these ribosomes depends on the source of the proteins, and not on the source of 23 S RNA.     

A Mechanistic Study of Au(III) Removal from Solution by Bacillus subtilis

Bacteria–Au interactions control the fate of Au in a variety of geologic systems. Although previous studies have determined that non-metabolizing Bacillus subtilis cells can remove Au(III) from solution via cell surface adsorption reactions, and that upon removal Au(III) is rapidly reduced to Au(I) and remains bound to the cell surface, the mechanism of Au(III) removal by B. subtilis is poorly understood. This study provides further constraints on the mechanisms responsible for Au(III) removal by B. subtilis by conducting batch Au(III) removal experiments as a function of pH and Au loading (Au:biomass ratio) using biomass with and without two different types of treatment: (1) a treatment to remove extracellular polymeric substances (EPS) from the biomass, and (2) a treatment to irreversibly block surface sulfhydryl sites from Au binding. The experimental results suggest that Au(III) removal can be attributed primarily to Au complexation with bacterial sulfhydryl sites, but that Au–amino binding is also important under some conditions. Our experiments also suggest that Au–sulfhydryl binding occurs predominantly on EPS molecules produced by B. subtilis, and that Au–amino binding is also important and is located within the bacterial cell envelope. These findings are the first to constrain the location of sulfhydryl-binding sites for B. subtilis biomass, and they are the first to demonstrate the important role played by bacterial EPS in the process of Au adsorption and reduction by bacteria.     

Photoreactivation in the genus Bacillus

Photoreactivation of ultraviolet radiation-induced DNA damage was examined in exponential-phase cells of six mesophilic species of the genus Bacillus. Under the experimental conditions used, it was observed that the laboratory strains B. cereus strain T and B. thuringiensis var. thuringiensis strain NRRL-B4039 exhibited strong photoreactivation (86-fold and 70-fold respectively). Bacillus licheniformis strain ATCC 8480 exhibited moderate (15-fold) photoreactivation. Weak photoreactivation was observed in B. subtilis strain 168 (4-fold) and B. megaterium strain QM B1551 (3.4-fold). Bacillus amyloliquefaciens strain H demonstrated no detectable photoreactivation.     

Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences

Partial gyrA sequences were determined for twelve strains belonging to Bacillus amyloliquefaciens, B. atrophaeus, B. licheniformis, B. mojavensis,B. subtilis subsp. subtilis, B. subtilissubsp. spizizenii and B. vallismortis. The average nucleotide and translated amino acid similarities for the seven type strains were 83.7 and 95.1%, respectively, whereas the corresponding value for the 16S rRNA sequences was 99.1%. All of the type strains were sharply separated; the closest relationship was found between B. atrophaeus and B. mojavensis which shared a nucleotide similarity of 95.8%. Phylogenetic trees were inferred from gyrA nucleotide sequences using the neighbor-joining, Fitch–Margoliash and maximum parsimony algorithms. The test strains were divided into four groups, which generally reflected results previously reported in restriction digest and DNA-DNA hybridization studies. It is concluded from the comparative sequence analysis that the gyrA sequences provide a firm framework for the rapid and accurate classification and identification of Bacillus subtilis and related taxa.     

Bactericidal Effect of ADP and Acetic Acid on Bacillus subtilis

Bacillus subtilis is a ubiquitous soil bacterium used for measuring the β-lysin activity and in other bioassays. We observed a complete bactericidal effect of ADP on B. subtilis at concentrations of 50–100 μM at pH values <5.5, which disappeared at pH values above 6. The effect was also found for acetic acid at concentrations >17.4 μM and similar pH values. ATP, adenosine, and HCl were not bactericidal. We used BCECF-AM, a pH-sensitive probe, and found that the killing of B. subtilis was due to a change in the intracellular pH caused by the passage across the cell membrane of these weak organic acids when incubated with B. subtilis at pH values near the pK. More experiments are needed to determine the biological meaning of these in vitro findings. Received: 14 June 1996 / Accepted: 19 July 1996