Biodegradation of saline phenolic wastewater in a biological contact oxidation reactor with immobilized cells of <Emphasis Type="Italic">Oceanimonas</Emphasis> sp.

Objective

To develop a method to treat saline phenolic wastewater in a biological contact oxidation reactor (BCOR) with immobilized cells of a marine microorganism, Oceanimonas sp., isolated from seawater.

Results

Cells were immobilized on fibre carriers in the BCOR. Saline wastewater with phenol at 1.5 g/l and NaCl at 6 % (w/v) was treated. In continuous assays, 99 % removal of phenol was achieved and a kinetic model for the phenol degradation is presented based on Monod’s equation.

Conclusion

The BOCR system using immobilized cells of Oceanimonas efficiently treats saline phenolic wastewaters without having decrease the salinity of the wastewater.

     

Degradation of phenol by a halotolerant strain of Penicillium chrysogenum

The lowest 50% lethal (effective) concentration, L(E)C₅₀, of phenol in a battery of seven microbiotests with species representing different trophic levels was 1–10 mg l⁻¹, classifying it as “toxic”. A phenol-degrading microorganism was isolated from soil samples of the salt mine of Clona in Portugal, after enrichment in the presence of phenol and high salt concentration. Based on cultural and morphological characteristics, the strain CLONA2 was identified as belonging to Penicillium chrysogenum. It was found to be a halotolerant fungus able to grow in a nutrient-rich medium with 5.8% NaCl. It degraded at least 300 mg l⁻¹ phenol as sole source of carbon and energy, without accumulation of intermediates. The samples were also tested for toxicity using the Microtox^® assay. Data showed that P. chrysogenum CLONA2 could be effectively utilized to reduce phenol toxicity. The results suggest also that phenol under saline conditions can be successfully mineralized by P. chrysogenum CLONA2.     

Characterization of levan produced by Serratia sp.

Levan was produced by a newly isolated bacterium from soil, taxonomically identified as a Serratia sp. This is the first report of levan production by Serratia sp. The levan was digested by levanase, which cannot hydrolyze β-2,1 linkages and the remaining substrate was analyzed by NMR. It was found that this levan had less β-2,1 linkage than other microbial levans, and that the structure was quite different from the levan produced by other bacteria such as genus Bacillus.     

Phylogeny of Bacteria Isolated from Rhabditis sp. (Nematoda) and Identification of Novel Entomopathogenic Serratia marcescens Strains

Twenty-five bacterial strains isolated from entomopathogenic nematodes were characterized to the genus level by 16S rRNA phylogeny and BLAST analyses. Bacteria strains isolated could be affiliated with seven genera. Microbacterium-like isolates phylogenetically affiliated with M. oxydans while those of Serratia were highly similar to S. marcescens. 16S rRNA sequences of Bacillus isolates matched those of both B. mycoides and B. weihenstephanesis. One isolate each matched Pseudomonas mosselii, Rheinheimera aquimaris, Achromobacter marplatensis, or Staphylococcus hominis. Serratia isolates were examined further for their pathogenicity to Galleria mellonella larvae. All the Serratia isolates exhibited potent pathogenicity toward G. mellonella larvae and possessed a metalloprotease gene encoding for a novel serralysin-like protein. The nucleotide sequence of the metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions when compared to the closest genBank entry, S. marcescens E-15, with an insertion of a new aspartic acid residue. Tajima’s test for equality of evolutionary rate was significant between the metalloprotease gene sequence of S. marcescens strain DOAB 216-82 (this study) and strain E-15. This new insecticidal metalloprotease gene and/or its product could have applications in agricultural biotechnology.     

Enterobacteriaceae and Pseudomonas aeruginosa Recovered from Vegetable Salads

Klebsiella, Enterobacter, and Serratia were recovered frequently in high counts from vegetable salads. Pseudomonas aeruginosa, although isolated frequently, was in lower counts.     

Slugs: Potential Novel Vectors of Escherichia coli O157

Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157.     

Rhizoremediation of phenol and chromium by the synergistic combination of a native bacterial strain and Brassica napus hairy roots

A bacterial strain resistant to phenol and Cr (VI) was isolated from an industrial polluted soil of Córdoba province (Argentina), which was identified as Pantoea sp. FC 1. This microorganism was able to use phenol as sole carbon source. In addition it was capable of reducing Cr (VI) to Cr (III) in mineral and nutrient media. The isolated strain exhibited some properties as plant-growth promoting bacterium (PGPB), such as production of Indole Acetic Acid (IAA) and synthesis of siderophores, as well as being capable of solubilizing inorganic phosphates. A rhizoremediation system using the association Pantoea sp. FC 1-Brassica napus hairy roots (HRs) was tested for phenol and Cr (VI) removal in a hydroponic system. Microbial inoculation improved both phenol removal and chromium accumulation efficiency by HRs, showing a significant increase in Cr (III) accumulation compared to non-inoculated HRs, exceeding 1000 mg kg⁻¹. Cr (III) was detected in HR biomass and supernatants, suggesting a possible Cr (VI) reducing activity of B. napus HRs. Basic studies in plant model systems, such as HRs, provide additional useful information that could facilitate the transition of this technology into plants suitable for practical rhizoremediation applications.     

Evaluation of insecticidal activity of a bacterial strain, <Emphasis Type="Italic">Serratia</Emphasis> sp. EML-SE1 against diamondback moth

To identify novel bioinsecticidal agents, a bacterial strain, Serratia sp. EML-SE1, was isolated from a dead larva of the lepidopteran diamondback moth (Plutella xylostella) collected from a cabbage field in Korea. In this study, the insecticidal activity of liquid cultures in Luria-Bertani broth (LBB) and nutrient broth (NB) of a bacterial strain, Serratia sp. EML-SE1 against thirty 3rd and 4th instar larvae of the diamondback moth was investigated on a Chinese cabbage leaf housed in a round plastic cage (Ø 10×6 cm). 72 h after spraying the cabbage leaf with LBB and NB cultures containing the bacterial strain, the mortalities of the larvae were determined to be 91.7% and 88.3%, respectively. In addition, the insecticidal activity on potted cabbage containing 14 leaves in a growth cage (165×83×124 cm) was found to be similar to that of the plastic cage experiment. The results of this study provided valuable information on the insecticidal activity of the liquid culture of a Serratia species against the diamondback moth.     

Study of phenol biodegradation using Bacillus amyloliquefaciens strain WJDB-1 immobilized in alginate–chitosan–alginate (ACA) microcapsules by electrochemical method

An aerobic microorganism with an ability to utilize phenol as sole carbon and energy source was isolated from phenol-contaminated wastewater samples. The isolate was identified as Bacillus amyloliquefaciens strain WJDB-1 based on morphological, physiological, and biochemical characteristics, and 16S rDNA sequence analysis. Strain WJDB-1 immobilized in alginate–chitosan–alginate (ACA) microcapsules could degrade 200 mg/l phenol completely within 36 h. The concentration of phenol was determined using differential pulse voltammetry (DPV) at glassy carbon electrode (GCE) with a linear relationship between peak current and phenol concentration ranging from 2.0 to 20.0 mg/l. Cells immobilized in ACA microcapsules were found to be superior to the free suspended ones in terms of improving the tolerance to the environmental loadings. The optimal conditions to prepare microcapsules for achieving higher phenol degradation rate were investigated by changing the concentrations of sodium alginate, calcium chloride, and chitosan. Furthermore, the efficiency of phenol degradation was optimized by adjusting various processing parameters, such as the number of microcapsules, pH value, temperature, and the initial concentration of phenol. This microorganism has the potential for the efficient treatment of organic pollutants in wastewater.     

Metabolism of 4-chloro-2-methylphenoxyacetic Acid by soil bacteria

A microorganism capable of degrading 4-chloro-2-methylphenoxyacetic acid (MCPA) was isolated from soil and identified as Flavobacterium peregrinum. All of the chlorine of MCPA was released as chloride, and the carboxyl-carbon was converted to volatile products by growing cultures of the bacterium, but a phenol accumulated in the medium. The phenol was identified as 4-chloro-2-methylphenol on the basis of its gas chromatographic and infrared characteristics. Extracts of cells of F. peregrinum and of a phenoxyacetate-metabolizing Arthrobacter sp. dehalogenated MCPA and several catechols but not 4-chloro-2-methylanisole. The Arthrobacter sp. cell extract was fractionated, and an enzyme preparation was obtained which catalyzed the conversion of MCPA to 4-chloro-2-methylphenol. The latter compound was not metabolized unless reduced nicotinamide adenine dinucleotide phosphate was added to the fractionated extract. The phenol in turn was apparently oxidized to a catechol by components of the enzyme preparation.     

Comparison between entrapment methods for phenol removal and operation of bioreactor packed with co-entrapped activated carbon and Pseudomonas fluorescence KNU417

A microorganism capable of degrading phenol was isolated from crude oil contaminated soil and identified as Pseudomonas fluorescence. A porous polymer bead of polyvinyl alcohol (PVA) and Xanthan gum was found to be the best entrapment for phenol degradation in terms of bead shape (spherical form), bead strength, non-agglomeration, phenol degradation rate, and cell holding inside the bead. Activated carbon was co-immobilized with the microorganism in the bead, which readily adsorbed phenol to decrease initial phenol concentration. Due to the decreased phenol concentration, the cells needed shorter adaptation time after which the microorganism stably degraded phenol. When the bead containing microorganism with 1% of activated carbon was packed in a packed-bed bioreactor, the start-up period was shortened by 40 h and the removal efficiency of phenol during the period was increased by 28% than the case with only microorganism.     

A Newly Isolated Organic Solvent Tolerant Staphylococcus saprophyticus M36 Produced Organic Solvent-Stable Lipase

Thirty-eight high lipase activity strains were isolated from soil, seawater, and Brassica napus. Among them, a novel organic solvent tolerant bacterium (strain M36) was isolated from the seawater in Jiangsu, China. Isolate M36 was able to grow at high concentration of benzene or toluene up to 40% (vol/vol), and later identified as Staphylococcus saprophyticus by biochemical test and 16s ribosomal DNA sequence. No work on Staphylococcus producing lipase with organic solvent tolerance has been reported so far. The lipase of strain M36 whose activity in liquid medium was 42 U mL⁻¹ at 24-h incubation time was stable in the presence of 25% (vol/vol) p-xylene, benzene, toluene, and hexane.     

Comparative Genome Analyses of Serratia marcescens FS14 Reveals Its High Antagonistic Potential

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.     

Isolation and Characterization of a Novel Thraustochytrid-like Microorganism that Efficiently Produces Docosahexaenoic Acid

A thraustochytrid-like microorganism (strain 12B) was isolated from the mangrove area of Okinawa, Japan. On the basis of its ectoplasmic net structure and biflagellate zoospores we determined strain 12B to be a novel member of the phylum Labyrinthulomycota in the kingdom Protoctista. When grown on glucose/seawater at 28 °C, it had a lipid content of 58% with docosahexaenoic acid (DHA; 22:6 n−3) at 43% of the total fatty acids. It had a growth rate of 0.38 h⁻¹. The DHA production rate of 2.8 ± 0.7 g l⁻¹ day⁻¹ is the highest value reported for any microorganism. Received 7 October 2005; Revisions requested 7 October 2005; Revisions received 15 November 2005; Accepted 15 November 2005     

Virulence of Serratia Strains against Costelytra zealandica

Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.     

Accelerated decolorization of reactive azo dyes under saline conditions by bacteria isolated from Arabian seawater sediment

Presence of huge amount of salts in the wastewater of textile dyeing industry is one of the major limiting factors in the development of an effective biotreatment system for the removal of azo dyes from textile effluents. Bacterial spp. capable of thriving under high salt conditions could be employed for the treatment of saline dye-contaminated textile wastewaters. The present study was aimed at isolating the most efficient bacterial strains capable of decolorizing azo dyes under high saline conditions. Fifty-eight bacterial strains were isolated from seawater, seawater sediment, and saline soil, using mineral salt medium enriched with 100?mg?l^?1 Reactive Black-5 azo dye and 50?g NaCl l^?1 salt concentration. Bacterial strains KS23 (Psychrobacter alimentarius) and KS26 (Staphylococcus equorum) isolated from seawater sediment were able to decolorize three reactive dyes including Reactive Black 5, Reactive Golden Ovifix, and Reactive Blue BRS very efficiently in liquid medium over a wide range of salt concentration (0–100?g NaCl l^?1). Time required for complete decolorization of 100?mg dye l^?1 varied with the type of dye and salt concentration. In general, there was an inverse linear relationship between the velocity of the decolorization reaction (V) and salt concentration. This study suggested that bacteria isolated from saline conditions such as seawater sediment could be used in designing a bioreactor for the treatment of textile effluent containing high concentration of salts.     

Genetic Dissection of Anopheles gambiae Gut Epithelial Responses to Serratia marcescens

Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component.     

Group-Specific Monitoring of Phenol Hydroxylase Genes for a Functional Assessment of Phenol-Stimulated Trichloroethylene Bioremediation

The sequences of the largest subunit of bacterial multicomponent phenol hydroxylases (LmPHs) were compared. It was found that LmPHs formed three phylogenetic groups, I, II, and III, corresponding to three previously reported kinetic groups, low-K_s (the half-saturation constant in Haldane's equation for trichloroethylene [TCE]), moderate-K_s, and high-K_s groups. Consensus sequences and specific amino acid residues for each group of LmPH were found, which facilitated the design of universal and group-specific PCR primers. PCR-mediated approaches using these primers were applied to analyze phenol/TCE-degrading populations in TCE-contaminated aquifer soil. It was found that the aquifer soil harbored diverse genotypes of LmPH, and the group-specific primers successfully amplified LmPH fragments affiliated with each of the three groups. Analyses of phenol-degrading bacteria isolated from the aquifer soil confirmed the correlation between genotype and phenotype. Competitive PCR assays were used to quantify LmPHs belonging to each group during the enrichment of phenol/TCE-degrading bacteria from the aquifer soil. We found that an enrichment culture established by batch phenol feeding expressed low TCE-degrading activity at a TCE concentration relevant to the contaminated aquifer (e.g., 0.5 mg liter⁻¹); group II and III LmPHs were predominant in this batch enrichment. In contrast, group I LmPHs overgrew an enrichment culture when phenol was fed continuously. This enrichment expressed unexpectedly high TCE-degrading activity that was comparable to the activity expressed by a pure culture of Methylosinus trichosporium OB3b. These results demonstrate the utility of the group-specific monitoring of LmPH genes in phenol-stimulated TCE bioremediation. It is also suggested that phenol biostimulation could become a powerful TCE bioremediation strategy when bacteria possessing group I LmPHs are selectively stimulated.