Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores

Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2′-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80 °C, and for the decolorization of indigo carmine at pH 8.0 and 60 °C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2 h at 37 °C. The apparent K_m of the enzyme displayed on spores was 443 ± 124 μM for ABTS with a V_max of 150 ± 16 U/mg spores. Notably, 1 mg of spores displaying B. subtilis laccase (3.4 × 10² U for ABTS as a substrate) decolorized 44.6 μg indigo carmine in 2 h. The spore reactor (0.5 g of spores corresponding to 1.7 × 10⁵ U in 50 mL) in a consecutive batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h at pH 8.0 and 60 °C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent.     

Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR

American foulbrood (AFB), a severe bacterial disease of honeybee brood, has recently been found in Uruguayan apiaries. Detection of the causative agent, Paenibacillus larvae subspecies larvae, is a very important concern in order to prevent disease dissemination and decrease of honey production. Since spores are the infective forms of this pathogen, in the present work we report the use of polymerase chain reaction (PCR) to detect P. l. subsp. larvae spores from in vitro cultures, larvae with clinical symptoms and experimentally contaminated honey. The set of primers was designed based on the published P. l. subsp. larvae 16S rRNA gene. Using this approach we could amplify the pathogen DNA and obtain a great sensitivity and a notable specificity. Detection limit for spore suspension was a 10^–2 dilution of template DNA obtained from 32 spores, as determined by plate count. For artificially contaminated honey, we could detect the PCR product at a 10^–3 dilution of template DNA obtained from 170 spores. In addition, when PCR conditions were set to improve specificity, we were able to amplify P. l. subsp. larvae DNA selectively and no cross-reactions were observed with a variety of related bacterial species, including P. l. subsp. pulvifaciens. Since spore detection is very important to confirm the presence of the disease, this method provides a reliable diagnosis of AFB from infected larvae and contaminated honey in a few hours.     

Decolorization of structurally different textile dyes by Aspergillus niger SA1

The fungal strain, Aspergillus niger SA1, isolated from textile wastewater sludge was screened for its decolorization ability for four different textile dyes. It was initially adapted to higher concentration of dyes (10–1,000 mg l⁻¹) on solid culture medium after repeated sub-culturing. Maximum resistant level (mg l⁻¹) sustained by fungal strain against four dyes was in order of; Acid red 151 (850) > Orange II (650) > Drimarene blue K₂RL (550) > Sulfur black (500). The apparent dye removal for dyes was seen largely due to biosorption/bioadsorption into/onto the fungal biomass. Decolorization of Acid red 151, Orange II, Sulfur black and Drimarine blue K₂RL was 68.64 and 66.72, 43.23 and 44.52, 21.74 and 28.18, 39.45 and 9.33% in two different liquid media under static condition, whereas, it was 67.26, 78.08, 45.83 and 13.74% with 1.40, 1.73, 5.16 and 1.87 mg l⁻¹ of biomass production under shaking conditions respectively in 8 days. The residual amount (mg l⁻¹) of the three products (α-naphthol, sulfanilic acid and aniline) kept quite low i.e., ≤2 in case AR 151 and Or II under shaking conditions. Results clearly elucidated the role of Aspergillus niger SA1 in decolorizing/degrading structurally different dyes into basic constituents.     

Decolorization of textile indigo dye by ligninolytic fungi

The indigo dye is extensively used by textile industries and is considered a recalcitrant substance, which causes environmental concern. Chemical products used on textile processing, which affect the environment through effluents, can be voluminous, colored and varied. Vat textile dyes, like indigo, are often used and dye mainly cellulosic fibers of cotton. Decolorization of this dye in liquid medium was tested with ligninolytic basidiomycete fungi from Brazil. Decolorization started in a few hours and after 4 days the removal of dye by Phellinus gilvus culture was in 100%, by Pleurotus sajor-caju 94%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No color decrease was observed in a sterile control. Thin layer chromatography of fungi culture extracts revealed only one unknown metabolite of Rf=0.60, as a result of dye degradation.     

Distribution of Paenibacillus larvae spores inside honey bee colonies and its relevance for diagnosis

One of the most important factors affecting the development of honey bee colonies is infectious diseases such as American foulbrood (AFB) caused by the spore forming Gram-positive bacterium Paenibacillus larvae. Colony inspections for AFB clinical symptoms are time consuming. Moreover, diseased cells in the early stages of the infection may easily be overlooked. In this study, we investigated whether it is possible to determine the sanitary status of a colony based on analyses of different materials collected from the hive. We analysed 237 bee samples and 67 honey samples originating from 71 colonies situated in 13 apiaries with clinical AFB occurrences. We tested whether a difference in spore load among bees inside the whole hive exists and which sample material related to its location inside the hive was the most appropriate for an early AFB diagnosis based on the culture method. Results indicated that diagnostics based on analysis of honey samples and bees collected at the hive entrance are of limited value as only 86% and 83%, respectively, of samples from AFB-symptomatic colonies were positive. Analysis of bee samples collected from the brood nest, honey chamber, and edge frame allowed the detection of all colonies showing AFB clinical symptoms. Microbiological analysis showed that more than one quarter of samples collected from colonies without AFB clinical symptoms were positive for P. larvae. Based on these results, we recommend investigating colonies by testing bee samples from the brood nest, edge frame or honey chamber for P. larvae spores.     

L-Tenuazonic Acid,a New Inhibitor of Paenibacillus Larvae

A search for bioactive compounds, inhibitors of Paenibacillus larvae, the causal agent of American foulbrood, a honeybees' disease, was carried on. Extracts of two fungal strains, Alternaria brassicicola and Alternaria raphani, isolated from pollen collected from beehives, exhibited a specific inhibitory activity against this bacterium. From these extracts and by means of chromatographic steps and bioassay-guided fractionation, three tetramic acids were isolated. The compounds were identified by spectroscopic methods and the absolute stereochemistry was chemically determined. L-Tenuazonic acid was shown to be responsible for the antibiotic activity. This compound showed a MIC of 32 μg/ml, comparable with that of oxytetracycline, an antibiotic currently used for the prevention of American foulbrood. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Decolorization of 1-aminoanthraquinone-2-sulfonic acid by Sphingomonas xenophaga

Sphingomonas xenophaga QYY from sludge samples could effectively decolorize 1-aminoanthraquinone-2-sulfonic acid (ASA-2), one kind of anthraquinone dye intermediate, under aerobic conditions. More than 98% of ASA-2 could be removed within 120 h at the dye concentration from 200 mg l⁻¹ to 1,000 mg l⁻¹ due to oxidative degradation. The strain converted ASA-2 to 2-(2′-hydroxy-3′-amino-4′-sulfo-benzoyl)-benzoic acid, 2-(2′-amino-3′-sulfo-6′-hydroxy-benzoyl)-benzoic acid, o-phthalic acid and 2-amino-3-hydroxy-benzenesulfonic acid, which were identified using HPLC-MS and NMR. A possible initial decolorization pathway was proposed according to these metabolites. The decolorization of ASA-2 by cells in the basal salt medium was induced by ASA-2, and was due to soluble cytosolic enzymes. Combined initial decolorization pathway and the analysis of decolorization enzyme(s), the major enzyme responsible for ASA-2 decolorization was a NADH-dependent oxygenase.     

Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB)

A novel micro-PCR-based detection method, termed ultra-rapid real-time PCR, was applied to the development of a rapid detection for Paenibacillus larvae (P. larvae) which is the causative agent of American Foulbrood (AFB). This method was designed to detect the 16S rRNA gene ofP. larvae with a micro-scale chip-based real-time PCR system, GenSpector^® TMC-1000, which has uncommonly fast heating and cooling rates (10 °C per second) and small reaction volume (6 μl). In the application of ultra-rapid real-time PCR detection to an AFB-infected larva, the minimum detection time was 7 min and 54 s total reaction time (30 cycles), including the melting temperature analysis. To the best of our knowledge, this novel detection method is one of the most rapid real-time PCR-based detection tools.     

Hydrogen production and anaerobic decolorization of wastewater containing Reactive Blue 4 by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa

Anaerobic biodegradability of wastewater (3,000 mg CODcr/l) containing 300 mg/l Reactive Blue 4, with different co-substrates, glucose, butyrate and propionate by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa, concomitantly with hydrogen production was investigated at 35°C. The accumulative hydrogen production at 3,067 mg CODcr/l was obtained after 7 days of incubation with glucose, sludge, the bacterial consortium. The volatile fatty acids, residual glucose and the total organic carbon were correlated to hydrogen obtained. Interestingly, the bacterial consortium possess decolorization ability showing approximately 24% dye removal after 24 h incubation using glucose as a co-substrate, which was about two and eight times those of butyrate (10%), propionate (12%) and control (3%), respectively. RB4 decolorization occurred through acidogenesis, as high volatile fatty acids but low methane was detected. The bacterial consortium will be the bacterial strains of interest for further decolorization and hydrogen production of industrial waste water.     

Identification of Paenibacillus larvae to the subspecies level: an obstacle for AFB diagnosis

This study was initially aimed at developing a PCR-test to differentiate between the pathogenic agent of American foulbrood (Paenibacillus larvae subsp. larvae) and powdery-scale disease (P. larvae subsp. pulvifaciens) of the honeybee. The test was based on the "insert of clone 9" (iC9), referring to a cloned 1.9 kB HaeIII fragment that occurs only in the P. larvae subsp. larvae reference strains and possibly correlates with American foulbrood virulence. It was shown that an iC9-based PCR-test discriminates between the BCCM/LMG reference strains of both subspecies. However, the screening of 179 Belgian field strains revealed five isolates that gave no iC9-based amplicon, thus rather resembling to P. larvae subsp. pulvifaciens. In addition, they all produced acid from mannitol, a characteristic previously assigned to the pulvifaciens subspecies. Because the reference strains gave conflicting data, this carbohydrate acidification was not conclusive. Therefore, the exact taxonomic position of the five retained strains was determined by a polyphasic approach using SDS-PAGE, AFLP, and ERIC-based PCR. Four iC9-negative field strains could be identified as P. larvae subsp. larvae; the taxonomic position of the fifth field strain remained ambiguous. The latter was provisionally classified as a subspecies pulvifaciens strain on the basis of SDS-PAGE. The present paper demonstrates the existence of field strains that do not fit well in the subdivision of the species P. larvae into two subspecies. Knowing that only one of both subspecies represents the pathogenic agent of AFB, this is a serious obstacle for the diagnosis of this honeybee disease.     

Short communication: Construction of a cloning vector for Paenibacillus polymyxa and P. azotofixans

A cloning vector that could replicate in Paenibacillus polymyxa, P. azotofixans and Bacillus subtilis was constructed using two Staphylococcus aureus plasmids. The recombinant plasmid confers chloramphenicol and erythromycin resistance and contains unique restriction sites for PvuII and BclI. The stability of pRJ45 was analysed.     

Characterization of Paenibacillus larvae bacteriophages and their genomic relationships to firmicute bacteriophages

Background

Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages.

Results

P. larvae phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 are myoviruses with ~50 kbp genomes. The six P. larvae phages form three distinct groups by dotplot analysis. An annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. Sixty phage assembly or structural protein genes and 133 regulatory or other non-structural protein genes were identifiable among the six P. larvae phages. Jimmer1, Jimmer2, and Davies formed stable lysogens resistant to superinfection by genetically similar phages. The correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages Emery and Abouo in electron micrographs. A Phamerator database was assembled with the P. larvae phage genomes and 107 genomes of Firmicute-infecting phages, including 71 Bacillus phages. Phamerator identified conserved domains in 1,501 of 6,181 phamilies (only 24.3%) encoded by genes in the database and revealed that P. larvae phage genomes shared at least one phamily with 72 of the 107 other phages. The phamily relationship of large terminase proteins was used to indicate putative DNA packaging strategies. Analyses from CoreGenes, Phamerator, and electron micrograph measurements indicated Jimmer1, Jimmer2, Abouo and Davies were related to phages phiC2, EJ-1, KC5a, and AQ113, which are small-genome myoviruses that infect Streptococcus, Lactobacillus, and Clostridium, respectively.

Conclusions

This paper represents the first comparison of phage genomes in the Paenibacillus genus and the first organization of P. larvae phages based on sequence and structure. This analysis provides an important contribution to the field of bacteriophage genomics by serving as a foundation on which to build an understanding of the natural predators of P. larvae.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-745) contains supplementary material, which is available to authorized users.     

Rapid biodegradation of metsulfuron-methyl by a soil fungus in pure cultures and soil

Summary Four strains of bacteria, 9 strains of fungi and 20 strains of actinomycetes capable of utilizing metsulfuron-methyl as sole carbon and energy source were isolated from a metsulfuron-methyl-treated soil by the enrichment culture method. A fungus named DS11F was selected as the most highly effective one according to the maximum tolerance concentration of 1,200 mg l⁻¹ and metsulfuron-methyl-degrading rate of 0.0716 g g⁻¹ cells h⁻¹, and was identified as an unknown strain of Penicillium sp. on the basis of colony growth, morphology and biochemical characteristics.␣Through liquid pure culture, the optimal metsulfuron-methyl-degrading conditions of DS11F were determined to be metsulfuron-methyl concentration 22.6 mg l⁻¹, inoculum concentration 12.25 mg l⁻¹, pH 7.0 and temperature 30°C. As additional C sources, supernatant of soaked compost could increase metsulfuron-methyl degradation by 8%, but glucose was ineffective. DS11F inoculation was found to significantly enhance the degradation of metsulfuron-methyl in soil, with the reduction of the concentration reaching 50% in 6 days. Admixture of compost could promote metsulfuron-methyl degradation to some extent. The growth of the inocula in the soils remained dominant and degradation resumed immediately when metsulfuron-methyl was applied again. The results show that addition of the isolated Penicillium sp. enhances the degradation of metsulfuron-methyl in water and soil.     

DNA extraction and PCR detection of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

Summary Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.     

Biosorption of Copper by Paenibacillus polymyxa Cells and their Exopolysaccharide

Summary Biosorption of heavy metals by gram-positive, non-pathogenic and non-toxicogenic Paenibacillus polymyxa P13 was evaluated. Copper was chosen as a model element because it is a pollutant originated from several industries. An EPS (exopolysaccharide)-producing phenotype exhibited significant Cu(II) biosorption capacity. Under optimal assay conditions (pH 6 and 25 °C), the adsorption isotherm for Cu(II) in aqueous solutions obeyed the Langmuir model. A high q value (biosorption capacity) was observed with whole cells (q_max=112 mgCu g⁻¹). EPS production was associated with hyperosmotic stress by high salt (1 M NaCl), which led to a significant increase in the biosorption capacity of whole cells (q_max=150 mgCu g⁻¹). Biosorption capacity for Cu(II) of the purified EPS was investigated. The maximum biosorption value (q) of 1602 mg g⁻¹ observed with purified EPS at 0.1 mg ml⁻¹ was particularly promising for use in field applications.     

Decolorization of Acid Orange 7 by bacteria of different tinctorial type: a comparative study

The abilities of two bacterial strains of opposite tinctorial type, the Gram-negative Alcaligenes faecalis and the Gram-positive Rhodococcus erythropolis, to decolorize reaction medium containing initially 10, 50, 100, 200 and 500 mg l⁻¹ of the monoazo dye Acid Orange 7 are discussed. The dye-binding properties of the strains and the starting rate of the decolorization reaction in dependence on the initial dye concentration are compared. An assumption is made that the higher dye-binding ability of A. faecalis is due to the existence of an outer membrane. The experimental data revealed relative independence of the decolorization dynamics on the dye-binding properties of the cell, which could be regarded as an indirect confirmation of the known extracellular redox-mediator-dependent mechanism of azo group reduction.     

Decolorization of Acid red 151 by Aspergillus niger SA1 under different physicochemical conditions

The fungal strain A. niger SA1 isolated from textile wastewater pond proved to be an important source of remediation (decolorization/degradation) for textile dye, AR 151 (Reactive diazo dye) under different physicochemical conditions. Decolorization assays of AR 151 were carried out in Simulated textile effluent under shake flask condition for 8 days. Decolorization (at 20 mg l⁻¹ of dye) and related biomass production overall decreased with increase in pH from 5 to 9, at 30°C. It was maximum (95.71%) at pH 5 with highest amount of three residual products (36.91 (α-naphthol = 5.72) (sulfanilic acid = 24.81) (aniline = 6.38)) besides 2.05 mg ml⁻¹ of biomass production at an optimum concentration 6 and 0.1 mg l⁻¹ of glucose and urea respectively. The formation of the three products followed a quite different pattern at different pH values, however, it was considerably low (Total = 2.81 mg l⁻¹) compared to the amount of decolorization (67.26%) at pH 8. Decolorization (95–97%) was most favored under mesophilic temperature (25–45°C). It increased i.e., 90–98% with subsequent increase in dye from 10 to 100 mg l⁻¹, kept ≥50% below 400 mg l⁻¹ and drastically declined to 17% at 500 mg l⁻¹ of dye. Apparently, decolorization is found to be associated with fungal growth and hyphal uptake mechanism (Biosorption/Bioadsorption), however, mineralization of AR 151 and related products under different operational conditions also suggested a metabolically mediated decolorization/degradation.     

Decolorization of textile dye by <Emphasis Type="Italic">Candida albicans</Emphasis> isolated from industrial effluents

The aim of the present work was to observe microbial decolorization and biodegradation of the Direct Violet 51 azo dye by Candida albicans isolated from industrial effluents and study the metabolites formed after degradation. C. albicans was used in the removal of the dye in order to further biosorption and biodegradation at different pH values in aqueous solutions. A comparative study of biodegradation analysis was carried out using UV–vis and FTIR spectroscopy, which revealed significant changes in peak positions when compared to the dye spectrum. Theses changes in dye structure appeared after 72 h at pH 2.50; after 240 h at pH 4.50; and after 280 h at pH 6.50, indicating the different by-products formed during the biodegradation process. Hence, the yeast C. albicans was able to remove the color substance, demonstrating a potential enzymatic capacity to modify the chemical structure of pigments found in industrial effluents.