Luria-Bertani Research Articles

Utilization of the acid hydrolysate of defatted Chlorella biomass as a sole fermentation substrate for the production of biosurfactant from Bacillus subtilis C9

Although algal biofuels have been promoted as a carbon-neutral alternative to fossil fuels, several economic and technological barriers challenge the much-anticipated success of algae as a commercial bio-feedstock. In particular, utilizing only a lipid portion of harvested algal biomass limits possible monetary returns from algal biomass within which non-lipid compounds generally comprise a major proportion. Previous studies thus demonstrated the production of ethanol from defatted algal biomass to improve overall economic outlook of the nascent algae industry; however, it is also possible to utilize residual algal biomass as a fermentation substrate for bacterial strains producing high value alternative products. Therefore, the goal of this study was to compare the yield of biosurfactant from B. subtilis C9 in conventional culture medium and the acid hydrolysate of defatted Chlorella biomass (DCB). Although increasing the concentration of acid catalyst from 0 N to 1.0 N was expected to proportionally enhance the effectiveness of dilute acid hydrolysis, the results indicated that DCB hydrolysates treated under different acid conditions at 121 °C for 15 min exhibited the highest yield of fermentable monosaccharides when treated with 0.25 N HCl. Subsequently, the fermentation of B. subtilis C9 was performed in both Luria-Bertani (LB) and DCB hydrolysate medium after adjusting salinity and monosaccharide concentration to identical levels; a significantly greater yield of biosurfactant was observed in the culture grown with DCB hydrolysate medium, while the presence of magnesium and manganese co-factors in DCB hydrolysate contributed to a greater biosurfactant yield. Collectively, these results suggest that the valorization of defatted algal biomass could be also accomplished by incorporating biosurfactant fermentation into algal biorefinery processes and call for further process optimization efforts that consider factors associated with the operation of industrial algal biorefineries.

Insecticidal Activity of Photorhabdus luminescens 0805-P2R Against Plutella xylostella.

Photorhabdus luminescens is an entomopathogenic rod-shaped bacterium infected with insect nematodes of the Heterorhabditidae family. It kills insects through the secretion of high molecular weight toxin complexes. In this study, Plutella xylostella larvae were orally administered P. luminescens for bioassay. After incubation in Luria-Bertani (LB) medium for a sufficiently long period, the mortality rates of P. xylostella observed after diluting the fermentation broth 50 times and diluting the supernatant 5 times were 18.89% and 91.11%, respectively. Retentates measuring more than 70kDa showed 88% mortality after ultrafiltration (UF) membrane treatment. Thus, the supernatant of P. luminescens had insecticidal activity, and the main insecticidal toxin complexes had a molecular weight exceeding 70kDa. The L9 (34) Taguchi orthogonal experimental optimized medium mode-predicted insecticidal activity levels were 84% and 119% in the 50-fold diluted fermentation broth and 5-fold diluted supernatant, respectively. Moreover, the insecticidal activity was improved to 92.2% in the 100-fold diluted fermentation broth and to 97.8% in the 10-fold diluted supernatant in the experiments. All combinations tested showed clear indications of lethality, including swelling, vesicle formation, cytoplasm vacuolization, and brush border membrane lysis. Thus, these results promote the use of P. luminescens 0805-P2R as a potent biopesticide to effectively control P. xylostella.

Innovative approach of in-situ fixed mode dual effect (photo-Fenton and photocatalysis) for ofloxacin degradation

Novel composite materials composed of clay, foundry sand (FS), and fly-ash (FA) have been employed to immobilize TiO2 for incorporating in-situ dual effect for the degradation of antibiotic ofloxacin. The in-situ generation of iron from the composite beads with surface active TiO2 induced the dual effect of photo-Fenton and photocatalysis. FA/FS/TiO2 beads illustrated the best results (92% removal) at optimized conditions in the batch reactor experiments. The increment in the rate constant along with a decrease in treatment time for the dual effect has proven the credentials of the in-situ dual effect. Synergy in first-order rate constant using dual process was 51% over the single processes of photo-Fenton and photocatalysis. After 35 recycles the viability of the composed beads was observed through SEM/EDS, UV-DRS and FT-IR analysis, which further justified its use industrially. Estimation of nitrate, nitrite, and ammonia as its by-products was performed for the confirmation of mineralization. Generation of the intermediate products was also identified through GC-MS analysis, and a degradation pathway was proposed. Toxicity test confirming the nontoxic nature of the treated solution was performed on E. coli grown in Miller’s Luria Bertani Broth nutrient medium.

Description of Azotobacter chroococcum subsp. isscasi subsp. nov. isolated from paddy soil and establishment of Azotobacter chroococcum subsp. chroococcum subsp. nov.

Three aerobic, asymbiotic, N2-fixing bacterial strains, designated P205T, P204 and P207, were isolated from a paddy soil in Yanting County, China. Based on 16S rRNA gene sequences, the three strains were closely related to Azotobacter chroococcum IAM 12666T (=ATCC 9043T) (99.00-99.79 % similarities). Strain P205T formed an individual branch distinct from the other two newly isolated strains and other related type strains in phylogenetic analyses based on 16S rRNA gene and 92 core genes. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values based on genome sequences of strain P205T and A. chroococcum ATCC 9043T, P204, P207 were near or slightly higher than the thresholds for species circumscription (95-96, 95-96 and 70 %, respectively), and the dDDH values were significantly lower than the threshold for delineating subspecies (79-80 %), which strongly supported that strain P205T belonged to A. chroococcum but was a novel subspecies distinct from the type strain of A. chroococcum. This finding was further corroborated by distinct phenotypic characteristics such as growth in Luria-Bertani (LB) medium, carbon source utilization and chemical sensitivity to vancomycin. Therefore, strain P205T represents a novel subspecies of Azotobacter chroococcum, for which the name Azotobacter chroococcum subsp. isscasi subsp. nov. is proposed with the type strain P205T (=KCTC 72233T=CGMCC 1.16846T=CCTCC AB 2019080T). The subspecies Azotobacter chroococcum subsp. chroococcum subsp. nov. is created automatically with the type strain ATCC 9043T (=DSM 2286T=JCM 20725T=JCM 21503T=LMG 8756T=NBRC 102613T=NCAIM B.01391T=NRRL B-14346T=VKM B-1616T).

Development of a Sensitive Escherichia coli Bioreporter Without Antibiotic Markers for Detecting Bioavailable Copper in Water Environments.

The whole-cell bioreporters based on the cop-operon sensing elements have been proven specifically useful in the assessment of bioavailable copper ions in water environments. In this study, a series of experiments was conducted to further improve the sensitivity and robustness of bioreporters. First, an Escherichia coli △copA△cueO△cusA mutant with three copper transport genes knocked out was constructed. Then, the copAp::gfpmut2 sensing element was inserted into the chromosome of E. coli △copA△cueO△cusA by gene knock-in method to obtain the bioreporter strain E. coli WMC-007. In optimized assay conditions, the linear detection range of Cu2+ was 0.025–5 mg/L (0.39–78.68 μM) after incubating E. coli WMC-007 in Luria–Bertani medium for 5 h. The limit of detection of Cu2+ was 0.0157 mg/L (0.25 μM). Moreover, fluorescence spectrometry and flow cytometry experiments showed more environmental robustness and lower background fluorescence signal than those of the sensor element based on plasmids. In addition, we found that the expression of GFPmut2 in E. coli WMC-007 was induced by free copper ions, rather than complex-bound copper, in a dose-dependent manner. Particularly, the addition of 40 mM 3-(N-Morpholino)propanesulfonic acid buffer to E. coli WMC-007 culture enabled accurate quantification of bioavailable copper content in aqueous solution samples within a pH range from 0.87 to 12.84. The copper recovery rate was about 95.88–113.40%. These results demonstrate potential applications of E. coli WMC-007 as a bioreporter to monitor copper contamination in acidic mine drainage, industrial wastewater, and drinking water. Since whole-cell bioreporters are relatively inexpensive and easy to operate, the combination of this method with other physicochemical techniques will in turn provide more specific information on the degree of toxicity in water environments.

Cyclic AMP-CRP Modulates the Cell Morphology of Klebsiella pneumoniae in High-Glucose Environment.

Bacteria can modify their morphology in response to environmental stimuli for survival or host defense evasion. The rich glucose in vivo or in the Luria–Bertani (LB) medium shortened the cell length of Klebsiella pneumoniae. The environmental glucose decreased the levels of cyclic AMP (cAMP) and the transcription of crp, which declined the cAMP–cAMP receptor protein (cAMP-CRP) activity. The cell length of crp deletion mutant was significantly shorter than that of the wild type (0.981 ± 0.057 μm vs. 2.415 ± 0.075 μm, P < 0.001). These results indicated that the high environmental glucose alters the bacterial morphology to a round form through regulating the activity of cAMP-CRP complex. Comparative proteomics analysis showed increased expression of 10 proteins involved in cell division or cell wall biosynthesis in the crp deletion strain. Five of them (ompA, tolB, ybgC, ftsI, and rcsF) were selected to verify their expression in the high-glucose environment, and overexpression of tolB or rcsF shortened the bacterial length similar to that of the crp deletion strain. Electrophoretic mobility shift assay indicated that CRP directly negatively regulates the transcription of tolB and rcsF by binding to the promoter regions. This study first proved the role and partial regulation mechanism of CRP in altering cell morphology during infection and provided a theoretical basis for elucidating the mechanism in diabetes mellitus susceptible to K. pneumoniae.

Development of micropatterning polyimide films for enhanced antifouling and antibacterial properties

A commercial biomedical Polyimide (PI) film was topographically and chemically modified by generating micrometric periodic arrays of lines using Direct Laser Interference Patterning (DLIP) in order to improve antifouling and antibacterial properties. DLIP patterning was performed with periods from 1 μm to 10 μm. The physical modification of the surface was characterized by SEM, AFM and contact angle measurements and, the chemical composition of the ablated surfaces was analyzed by ATR-IR and XPS spectroscopies. The antibacterial effects were evaluated through the effect on Pseudomonas aeruginosa colonies growth on the LB (Luria Bertani) broth. The results showed that the laser treatment change the topography and as a consequence the chemistry surface, also that the microstructured surfaces with periods below 2 μm, exhibited a significant bacterial (P. aeruginosa) adhesion decrease compared with non-structured surfaces or with surfaces with periods higher than 2 μm. The results suggest that periodic topography only confer antifouling properties and reduction of the biofilm formation when the microstructure presents periods ranging from 1 μm to 2 μm. On the other hand, the topography that confer strong antifouling superficial properties persists at long incubation times. In that way, polymer applications in the biosciences field can be improved by a surface topography modification using a simple, single-step laser-assisted ablation method.

Isolation and Molecular Identification of Two Chitinase Producing Bacteria from Marine Shrimp Shell Wastes.

Chitinase enzymes have a various application in the field of environmental, biotechnology and medical aspects. This study aimed to the production of the chitinolytic enzymes from different species of bacteria. Bacterial isolation from different habitats was carried out on agar medium containing chitin as carbon and nitrogen sources. The obtained bacteria (20) were characterized and screened again in chitin broth medium. Out of 20 bacterial isolate, 2 new isolates, belonged to Streptomyces laurentii SN5 and Cellulosimicrobium funkei SN20, were the most active in chitin degradation compared to the other isolates. They have been characterized for the first time for their chitinase activity. They were identified using 16S rRNA gene analysis and in the liquid medium, the 2 isolates have enzyme activities of 0.533 and 0.537 U mL-1, respectively. The maximum chitinase production was obtained when those bacterial strains were grown in Luria-Bertani (LB) broth amended with 1% colloidal chitin, for 1 day and at temperature of 30°C. The optimum pH value for chitinase production was pH 7 for both S. laurentii and C. funkei. The enzyme has been purified using Sephadex G-100 and DEAE-Cellulose chromatography column and found to have a similar molecular size of ~50 kDa. Those two bacterial species could be used in chitinase production and in the environmental recycling of disposable chitin wastes such as chitin from shrimp shell waste.

First Report of Dickeya fangzhongdai Causing Soft Rot of Onion in New York State

In early July 2014, an outbreak of soft rot disease of onion (Allium cepa L.) occurred in Orange County, NY. Symptoms developed following rainy, hot, humid weather and included chlorosis, maceration of leaves, browning, and plant collapse within a few days. Bulbs from diseased plants appeared normal, but inner tissues were macerated. Disease was first apparent in a field of red onions (cv. Redwing); later yellow onions (cultivar unknown) in an adjacent field were symptomatic. Standard techniques were used to isolate bacteria from diseased red onion bulbs (Bonasera et al. 2017). Briefly, 14 onion bulbs were cut open with a disinfected knife, and diseased tissues were probed with sterile wooden applicators and streaked on onion extract medium agar (Zaid et al. 2012). Plates were incubated for 2 days at 28°C, and individual colonies were streaked to purity on Luria–Bertani (LB) agar. Small white bacterial colonies with irregular margins were obtained as the predominant type from three samples. A representative colony was identified as Dickeya sp. based on the sequence of a portion of its 16S rRNA gene amplified by fD1/rP2 (Weisburg et al. 1991). Several more colonies were assessed using ADE1 and ADE2 PCR primers specific to Dickeya genus (Nassar et al. 1996), which also indicated that these isolates belonged to the genus Dickeya. Genomic DNA of one of these colonies, AP6, was extracted and sequenced using an Illumina NextSeq500 by the Institute of Biotechnology at Cornell University. Raw reads were processed (Ma et al. 2019) and used for de novo assembly with SPAdes 1.12. The draft genome assembly resulted in 260 contigs, with an N50 size of 50,007 bp, totaling 5.11 Mbp, and it was deposited in GenBank as accession number VS. We conducted average nucleotide identity and in silico DNA-DNA hybridization tests to determine the species of Dickeya strain AP6 using Python package pyANI and GGDC (https://ggdc.dsmz.de), respectively. These analyses indicated that AP6 belongs to species Dickeya fangzhongdai. Koch’s postulates were completed with D. fangzhongdai AP6. Two leaves each of three 80-day-old, healthy-appearing, controlled environment-grown onion plants (cv. Fortress for the first replicate, cv. Redwing for the second and third replicates) were inoculated with D. fangzhongdai AP6. The leaves were first punctured with sterile toothpicks, and then the wound was smeared with bacteria grown overnight on LB agar. One plant was punched with sterile toothpicks as a negative control. Elongated lesions with water-soaking at the margins were observed around the inoculation sites after incubating for 24 h at 30°C; no symptoms were observed on control plants. Disease symptoms progressed very rapidly; after 72 h, all AP6-inoculated plants had collapsed completely, and foliage was quite water-soaked. The experiment was conducted three times. We isolated three isolates from water-soaked lesions 24 h after inoculation and used dnaX sequencing on one of the reisolated colonies to confirm that bacteria in the diseased tissue were identical to the D. fangzhongdai inoculum. D. fangzhongdai was first identified in China as causing bleeding canker necrosis on pear (Tian et al. 2016). D. fangzhongdai was reported to cause soft rot in Welsh onion in Taiwan (Tsai et al. 2019), diseased Phalaenopsis orchids in Slovenia and Aglaonema in St. Lucia, and was found in water samples from Scotland and Malaysia (Alic et al. 2018; Pritchard et al. 2013). Soft rot disease of onion was reported in the United States (Ceponis et al. 1986), but its causal organism had not been investigated thoroughly. Here we report that D. fangzhongdai caused an outbreak of soft rot of onions in Orange County, NY.

Optimization of Conditions for the Higher Level Production of Protease: Characterization of Protease from Geobacillus SBS-4S

Current study was planned keeping in mind the importance of proteases and their role in industry. In the present study protease was produced using Luria Bertani medium. The LB medium was supplemented with various carbon and nitrogen sources separately for the enhancement of growth of Geobacillus SBS-4S and for protease production. The optimization studies demonstrated the increase in protease production from 10.6 to 24.4 U/mL or 34.5 U/mL when the medium was supplemented with additional 2% yeast extract or 5% wheat bran respectively. Under the optimal conditions we could produce 46.2 U/mL of protease. The protein was purified by column chromatography and the purified protein was utilized for characterization studies. SDS-PAGE analysis confirmed the molecular weight of protease as 37 kDa. The enzyme exhibited its maximal activity at 60 °C and pH 9.0. Presence of Ca2+ and Mn2+ at a final concentration of I mM in the activity assay mixture enhanced protease activity from 100% to 133 and 150% respectively. Protease activity was slightly reduced (92%) in the presence of SDS whereas the presence of non-ionic detergents Triton X-100, Tween-20 and Tween-80 reduced the enzyme activity to 87, 82 and 69% respectively. Thermostability studies demonstrated that the protein was stable with 50% residual activity after an incubation of 2 h and 25 min at 60 °C in the presence of 1 mM Mn2+. The kinetic studies demonstrated the Km and Vmax values of 16.67 mg/mL and 143 U/mL respectively. The stability of protease at wide range of pH and temperature makes this enzyme suitable for its utilization in detergent and poultry feed industry.

Occurrence and Diversity of Intra- and Interhospital Drug-Resistant and Biofilm-Forming Acinetobacter baumannii and Pseudomonas aeruginosa.

Acinetobacter baumannii and Pseudomonas aeruginosa are the most relevant Gram-negative bacteria associated with hospital and opportunistic infections. This study aimed to evaluate the dynamics of drug-resistant A. baumannii and P. aeruginosa and biofilm formers from two public hospitals in northeastern Brazil. One hundred isolates (35 from A. baumannii and 65 from P. aeruginosa) were identified using the automated Vitek®2 Compact method (bioMérieux) and confirmed using the MALDI-TOF (MS) mass spectrometry technique. Molecular experiments were performed by polymerase chain reaction (PCR) to detect the frequency of blaKPC, blaIMP, blaVIM, and blaSHV genes. The biofilm formation potential was evaluated using crystal violet in Luria Bertani Miller and trypticase soy broth culture media under the following conditions: at standard concentration, one quarter (25%) of the standard concentration and supplemented with 1% glucose. In addition, the genetic diversity of the isolates was verified by the ERIC-PCR technique. Isolates presented distinct resistance profiles with a high level of beta-lactam resistance. The highest index of genes detected was blaKPC (60%), followed by blaSHV (39%), blaVIM (8%), and blaIMP (1%). All the isolates were sensitive to the polymyxins tested and formed biofilms at different intensities. Twelve clones of A. baumannii and eight of P. aeruginosa were identified, of which few were indicative of intra- and interhospital dissemination. This study reveals the dispersion dynamics of these isolates in the hospital environment. The results demonstrate the importance of monitoring programs to combat the spread of these pathogens.

Embedding ultrasmall Ag nanoclusters in Luria-Bertani extract via light irradiation for enhanced antibacterial activity

Ultrasmall silver nanoclusters (Ag NCs) with rich surface chemistry and good biocompatibility are promising in antibacterial application, however, further development of Ag NCs for practical settings has been constrained by their relatively weak antibacterial activity. Using the nutritionally-rich medium for bacteria (e.g., Luria-Bertani (LB) medium) to coat active Ag NCs could further improve their antibacterial activity. Here, we provide a delicate design of a highly efficient Ag NCs@ELB antibacterial agent (ELB denotes the extract of LB medium) by anchoring Ag NCs inside the ELB species via light irradiation. The as-designed Ag NCs with bacterium-favored nutrients on the surface can be easily swallowed by the bacteria, boosting the production of the intracellular reactive oxygen species (ROS, about 2-fold of that in the pristine Ag NCs). Subsequently, a higher concentration of ROS generated in Ag NCs@ELB leads to enhanced antibacterial activity, and enables to reduce the colony forming units (CFU) of both gram-positive and gram-negative bacteria with 3–4 orders of magnitude less than that treated with the pristine Ag NCs. In addition, the Ag NCs@ELB also shows good biocompatibility. This study suggests that surface engineering of active species (e.g., Ag NCs) with nutritionally-rich medium of the bacteria is an efficient way to improve their antibacterial activity.

Synthesis and antibacterial activity of iron manganite (FeMnO3) particles against the environmental bacterium Bacillus subtilis.

Nanocrystalline iron manganite powder was synthesized using the sol–gel combustion process, with glycine as fuel. It was further calcined at 900 °C for 8 h, resulting in the formation of a loose cubic FeMnO3 powder with a small specific surface area, net-like structure and plate-like particles as confirmed by XRD, N2 physisorption, FESEM and TEM analyses. The metal ion release was studied by ICP-OES and showed that less than 10 ppb of Fe or Mn ions were released by leaching in water, but 0.36 ppm Fe and 3.69 ppm Mn was found in LB (Luria–Bertani) bacterial medium. The generation of reactive oxygen species (ROS) was monitored in distilled water and bacterial medium and showed that FeMnO3 particles do not generate O2˙− ions with or without UV irradiation, but synthesize H2O2 and show an antioxidative effect. Besides the higher stability of FeMnO3 particles in aqueous solution they showed an inhibitory effect on Bacillus subtilis growth in LB medium even at low concentrations (0.01 mg ml−1), but not in BHI medium even at 1 mg ml−1. This study points out that the mechanism of antibacterial action of engineered metal oxides needs continued investigation and specific experimental controls.

Isolation of endophytic bacteria from Rehmannia glutinosa Libosch and their potential to promote plant growth.

In order to study the growth promoting potential of endophytic bacteria from Rehmannia glutinosa Libosch, a total of 25 different bacteria belonging to 7 genera were identified by 16S rRNA gene sequencing, including Bacillus, Micrococcus, Lysinibacillus, Brevibacterium, Halomonas, Kocuria and Terribacillus. In this study, thirteen bacterial strains were found to solubilize inorganic phosphate, with the isolate Kocuria rosea (EH15) having the highest phosphorus dissolution activity (3.70 μg/mL). Twelve isolates were positive for nitrogen fixation abilities. Twenty-two strains produced indole-3-acetic acid (IAA) in the presence of L-tryptophan, and eleven of the twenty-two isolates synthesized IAA in the absence of L-tryptophan. The strain K. rosea (EH15) was capable of producing the highest IAA amount (15.36 and 7.98 mg/L) in Luria Bertani (LB) broth containing 0.2% L-tryptophan and lacking L-tryptophan, respectively. Ten isolates had siderophore production abilities with Bacillus amyloliquefacieus EH10 (0.26) and Brevibacterium frigoritolerans EH13 (0.32) showing high siderophore production characteristics. Five bacteria endogenous were selected to evaluate the growth parameters of Brassica napus L. and all isolates exhibited a significantly greater increase in seedling height, root length, fresh weight and dry weight, than the control plants. The greatest improvement appeared in the case of co-inoculation of EH10 and EH15, except in dry weight, and the biggest enhancement in dry weight occurred in the strain EH15. In general, these endophytic bacteria indicate a potential as microbial fertilizers to promote the growth of R. glutinosa Libosch.

Biofilm Production Potential of Salmonella Serovars Isolated from Chickens in North West Province, South Africa.

Bacterial biofilms have recently gained considerable interest in the food production and medical industries due to their ability to resist destruction by disinfectants and other antimicrobials. Biofilms are extracellular polymer matrices that may enhance the survival of pathogens even when exposed to environmental stress. The effect of incubation temperatures (25°C, 37°C, and 40°C) and Salmonella serotype on biofilm-forming potentials was evaluated. Previously typed Salmonella serotypes (55) isolated from the gut of chickens were accessed for biofilms formation using a standard assay. Salmonella Typhimurium ATCC 14028TM and Salmonella Enteritidis ATCC 13076TM (positive controls), Escherichia coli (internal control) and un-inoculated Luria Bertani (LB) broth (negative control) were used. The isolates formed no biofilm (11.86–13.56%), weak (11.86–45.76%), moderate (18.64–20.34%), strong biofilms (23.73–54.24%) across the various temperatures investigated. Serotypes, Salmonella Heidelberg and Salmonella Weltevreden were the strongest biofilm formers at temperatures (25°C, 37°C, and 40°C, respectively). The potential of a large proportion (80%) of Salmonella serotypes to form biofilms increased with increasing incubation temperatures but decreased at 40°C. Findings indicate that average temperature favours biofilm formation by Salmonella serotypes. However, the influence of incubation temperature on biofilm formation was greater when compared to serotype. A positive correlation exists between Salmonella biofilm formed at 25°C, 37°C and 40°C (p ≥ 0.01). The ability of Salmonella species to form biofilms at 25°C and 37°C suggests that these serotypes may present severe challenges to food-processing and hospital facilities.

Biomineralization Performance of Bacillus sphaericus under the Action of Bacillus mucilaginosus

Microbial Induced Calcite Precipitation (MICP) is a biochemical process widely found in nature, also known as microbial mineralization. This paper investigates whether this process can help promote the intelligent reinforcement and repair of underground projects such as mines and tunnels. We selected Bacillus sphaericus and Bacillus mucilaginosus as the research objects. The former has an outstanding urease production ability, and the latter can secrete carbonic anhydrase in vitro. Bacillus mucilaginosus was introduced into the culture solution of Bacillus sphaericus in the most suitable culture environment, and the changes of mineralization rate and mineralization yield of Bacillus sphaericus were observed and analyzed. The results revealed that, to maintain the highest growth rate of Bacillus sphaericus, the optimal pH value was between 7 and 8, the optimal urea concentration was 0.5 mol/L, the optimal Ca2+ concentration was 0.6 mol/L, and the optimal Luria‐Bertani (LB) culture concentration was 20 g/L. The amount of biomineralized calcium carbonate precipitated in the double bacteria solution can reach 1.89 times the amount of the precipitation in the Bacillus sphaericus solution under the same conditions. It concludes that the introduction of Bacillus mucilaginosus can effectively increase the mineralization yield of Bacillus sphaericus without affecting the mineralized products.

Occurrence of Copper-Resistant Xanthomonas perforans and X. gardneri in Illinois Tomato Fields

This study was conducted to determine whether currently prevailing isolates of Xanthomonas perforans and X. gardneri, the causal agents of tomato bacterial spot in Illinois, were resistant to copper. First, 133 isolates from each pathogen were tested for their multiplication on mannitol glutamate yeast agar (MGYA) amended with 0.8 mM of laboratory-grade copper sulfate. Results showed that 59% of X. perforans and 38% of X. gardneri isolates formed colonies on the copper-amended culture. Then, six isolates of X. perforans and five isolates of X. gardneri were tested for their multiplication on MGYA amended with 0.2, 0.8, 1.2, 1.6, and 2.0 mM of laboratory-grade of either copper hydroxide or copper sulfate. All 11 isolates formed colonies on cultures with 1.2 mM or lower rates of either copper compound. Molecular analysis revealed the presence of the copper-resistance genes copA and copM in the isolates. In vitro assays showed that both X. perforans and X. gardneri formed colonies when grown on Luria–Bertani agar containing low concentrations of copper hydroxide (Kocide-3000 46.1DF) and copper sulfate pentahydrate (Instill). Double Nickel LC, a biopesticide containing Bacillus amyloliquefaciens strain D747, was the most effective in preventing multiplication of copper-resistant isolates of both pathogens on the culture media.

Isolation of Bacillus subtilis SJ4 from Saeu (Shrimp) Jeotgal, a Korean Fermented Seafood, and Its Fibrinolytic Activity

A Bacillus strain, SJ4, exhibiting strong fibrinolytic activity was isolated from saeu (shrimp, Acetes chinensis) jeotgal, a Korean traditional fermented food and was identified as B. subtilis. The B. subtilis SJ4 strain can grow at a NaCl concentration of up to 15% (w/v). The fibrinolytic activity of B. subtilis SJ4 (152.0 U/ml) cultured in Luria-Bertani (LB) broth for 48 h at 37℃ with aeration was higher than that of B. subtilis SJ4 cultured in TSB (124.5 U/ml) under same culture conditions. The major proteins in the LB culture supernatant of B. subtilis SJ4 were analyzed by SDS-PAGE, which revealed three major bands (23, 25, and 28 kDa). The band (23 kDa) with strong fibrinolytic activity, analyzed on fibrin zymogram, was observed at 60-96 h of cultivation. The aprESJ4 gene encoding the major fibrinolytic enzyme, AprESJ4, was cloned by PCR. The aprESJ4 gene sequence exhibited high similarities with the fibrinolytic gene sequences of other Bacillus species. The amino acid sequence of AprESJ4 exhibited 98.9 and 98.4% similarity with subtilisin NAT and AprE2 of B. subtilis, respectively. Hence, B. subtilis SJ4 can be a potential starter culture for jeotgal products.

Development of Selective Enrichment Medium for Clinical Isolates of Vibrio vulnificus Based upon Virulence Correlating Genes

The objective of this study was to find out if different enrichment media would influence the selection of clinical and environmental strains of Vibrio vulnificus. Therefore, traditional enrichment and maintenance medium for V. vulnificus were tested for selective preference for clinical or environmental strains. In the laboratory, Alkaline Peptone Water (APW) and Brain Heart Infusion Broth (BHIB), with adjusted salinity to that of APW, were inoculated with equal concentrations of clinical and environmental strains and incubated for 20 hr at 35 °C. BHIB selected for clinical strains (85%) as indicated by percentage of vcgC to vvhA genes, enumerated with quantitative PCR. In APW, the ratio of clinical (47%) to environmental strains (53%) was roughly equal. Enrichments of seawater with BHIB, APW, and Luria Bertani broth (LB) resulted in higher percentage of clinical strains in BHIB but not in APW or LB, in most samples. However seasonal differences in predominance of clinical or environmental strains in the seawater samples influenced the enrichment process. Our study suggests that BHIB10 might be advantageous for studying virulence factors and APW10 could be suitable for isolation and MPN enumeration in V. vulnificus. The selective preference of the BHIB10 medium for clinical strains indicates that iron plays an important role in the infection of the human host.

Recovery of Metals from Acid Mine Drainage by Bioelectrochemical System Inoculated with a Novel Exoelectrogen, Pseudomonas sp. E8.

Acid mine drainage (AMD) is a typical source of environmental pollution ascribing to its characteristics of high acidity and heavy metal content. Currently, most strategies for AMD treatment merely focus on metal removal rather than metal recovery. However, bioelectrochemical system (BES) is a promising technology to simultaneously remove and recover metal ions from AMD. In this study, both cupric ion and cadmium ion in simulated AMD were effectively recovered by BES inoculated with a novel exoelectrogen, Pseudomonas sp. E8, that was first isolated from the anodic electroactive biofilm of a microbial fuel cell (MFC) in this study. Pseudomonas sp. E8 is a facultative anaerobic bacterium with a rod shape, 0.43–0.47 μm wide, and 1.10–1.30 μm long. Pseudomonas sp. E8 can agglomerate on the anode surface to form a biofilm in the single-chamber MFC using diluted Luria-Bertani (LB) medium as an energy substrate. A single-chamber MFC containing the electroactive Pseudomonas sp. E8 biofilms has a maximum output voltage of 191 mV and a maximum power density of 70.40 mW/m2, which is much higher than those obtained by most other exoelectrogenic strains in the genus of Pseudomonas. Almost all the Cu2+ (99.95% ± 0.09%) and Cd2+ (99.86% ± 0.04%) in simulated AMD were selectively recovered by a microbial fuel cell (MFC) and a microbial electrolysis cell (MEC). After the treatment with BES, the high concentrations of Cu2+(184.78 mg/L), Cd2+(132.25 mg/L), and total iron (49.87 mg/L) in simulated AMD were decreased to 0.02, 0.19, and 0 mg/L, respectively. Scanning electron micrograph (SEM), energy dispersive X-ray spectrometry (EDXS) and X-ray diffraction (XRD) analysis indicate that the Cu2+ and Cd2+ in simulated AMD were selectively recovered by microbial electrochemical reduction as Cu0 (together with trace amounts of Cu2O) or Cd0 on the cathode surface. Collectively, data suggest that Pseudomonas sp. E8 has great potential for AMD treatment and metal recovery.