Complete oxidation of linear alkylbenzene sulfonate by bacterial communities selected from coastal seawater.

Anionic surfactants, especially alkylbenzene sulfonates, are discharged into marine areas in great quantities. Because of their poor biodegradability, linear alkylbenzene sulfonates accumulate in seawater and sediments. Bacterial communities that can degrade surfactants were selected from coastal seawater contaminated by urban sewage. All the isolated strains consisted of gram-negative, strictly aerobic rods or helical bacteria. Some of these, though isolated from coastal seawater, did not need sodium for growth and appeared to be related to the genera Alcaligenes and Pseudomonas. Complete surfactant biodegradation was achieved by three important steps: terminal oxidation of the alkyl chain, desulfonation, and aromatic-ring cleavage. Only a few strains were able to carry out the first two steps. The aromatic ring was then cleaved by other strains that possess very specific enzymatic activities. Finally, a number of strains grew on short acids that were end-of-metabolism products of the others.     

Characterization of a Vibrio cholerae phage isolated from the coastal water of Peru

A Vibrio cholerae bacteriophage, family Myoviridae, was isolated from seawater collected from the coastal water of Lima, Peru. Genome size was estimated to be 29 kbp. The temperate phage was specific to V. cholerae and infected 12/13 V. cholerae O1 strains and half of the four non-O1/non-O139 strains tested in this study. Vibrio cholerae O139 strains were resistant to infection and highest infection rates were obtained in low nutrient media amended with NaCl or prepared using seawater as diluent.     

Microbial Specificity of Metallic Surfaces Exposed to Ambient Seawater

High-molecular-weight materials associated with the extracellular matrix and film found on titanium and aluminum surfaces after exposure to flowing coastal seawater were isolated. This material was purified by hydroxylapatite chromatography and subsequently employed to produce antibodies in the toad, Bufo marinus. The antibodies were immobilized on a solid support and employed to isolate adhesion-enhancing, high-molecular-weight materials from the laboratory culture media of bacterial strains recovered from the respective metallic surfaces during the course of their exposure to seawater. The adhesion-enhancing materials produced by the surface-associated bacterial strains were immunologically related to the extracellular biofouling matrix material found on the surfaces from which these bacteria were isolated. The surface selectivity of these bacterial strains appeared to be based on the specificity of the interaction between adhesion-enhancing macromolecules produced by these bacteria and the surfaces in question.     

DNA microarray mediated transcriptional profiling of Nitrosomonas europaea in response to linear alkylbenzene sulfonates

Linear alkylbenzene sulfonates (LAS) constitute, quantitatively, the most important group of synthetic surfactants used today. We studied the gene expression of Nitrosomonas europaea in response to LAS using a DNA microarray because ammonia-oxidizers are thought to be more sensitive to LAS than other microorganisms. Our objective was to elucidate which genes are expressed for N. europaea in response to LAS exposure. Microarray analysis and real-time PCR assay revealed that c. 30 genes were significantly expressed after LAS exposure, in particular genes associated with energy production and conversion. Our findings demonstrate that physical disruption of membrane structures, which contain enzymes associated with energy production and conversion, might be an important explanation for the high sensitivity of N. europaea to LAS exposure.     

Pseudomonas C12B, an SDS degrading strain, harbours a plasmid coding for degradation of medium chain length n-alkanes

Pseudomonas C12B is able to degrade alkyl sulfates, alkylbenzene sulfonates, and linear alkanes and alkenes. Mitomycin C curing experiments and conjugation experiments demonstrated that the ability to utilize n-alkanes (C₉–C₁₂) and n-alkenes (C₁₀ and C₁₂) of medium chain length was plasmid-encoded. The plasmid was designated pDEC. Its size was estimated at several hundreds kb according to mobility in agarose gels. The plasmid did not confer resistance to the antibiotics tested. Analysis of alkylsulfatases P1 and P2 in original and cured strains confirmed that both enzymes are encoded by the chromosome. The ability of Pseudomonas C12B to utilize alkylbenzene sulfonates also appears to be encoded by the chromosome. pDEC could be transferred only to cured derivatives of Pseudomonas C12B, but not to strains of P. aeruginosa, P. putida, or Acinetobacter sp. Cured derivatives of Pseudomonas C12B could not serve as hosts for the broad host range plasmid CAM–OCT. The enzyme system encoded by the putative dec genes present on plasmid pDEC differs from the system coded by the alk genes of plasmid OCT in the size range of hydrocarbons preferentially used.     

Diversity and association of psychrophilic bacteria in Antarctic sea ice.

The bacterial populations associated with sea ice sampled from Antarctic coastal areas were investigated by use of a phenotypic approach and a phylogenetic approach based on genes encoding 16S rRNA (16S rDNA). The diversity of bacteria associated with sea ice was also compared with the bacterial diversity of seawater underlying sea ice. Psychrophilic (optimal growth temperature, < or = 15 degrees C; no growth occurring at 20 degrees C) bacterial diversity was found to be significantly enriched in sea ice samples possessing platelet and bottom ice diatom assemblages, with 2 to 9 distinct (average, 5.6 +/- 1.8) psychrophilic taxa isolated per sample. Substantially fewer psychrophilic isolates were recovered from ice cores with a low or negligible population of ice diatoms or from under-ice seawater samples (less than one distinct taxon isolated per sample). In addition, psychrophilic taxa that were isolated from under-ice seawater samples were in general phylogenetically distinct from psychrophilic taxa isolated from sea ice cores. The taxonomic distributions of psychrotrophic bacterial isolates (optimal growth temperature, > 20 degrees C; growth can occur at approximately 4 degrees C) isolated from sea ice cores and under-ice seawater were quite similar. Overall, bacterial isolates from Antarctic sea ice were found to belong to four phylogenetic groups, the alpha and gamma subdivisions of the Proteobacteria, the gram-positive branch, and the Flexibacter-Bacteroides-Cytophaga phylum. Most of the sea ice strains examined appeared to be novel taxa based on phylogenetic comparisons, with 45% of the strains being psychrophilic. 16S rDNA sequence analysis revealed that psychrophilic strains belonged to the genera Colwellia, Shewanella, Marinobacter, Planococcus, and novel phylogenetic lineages adjacent to Colwellia and Alteromonas and within the Flexibacter-Bacteroides-Cytophaga phylum. Psychrotrophic strains were found to be members of the genera Pseudoalteromonas, Psychrobacter, Halomonas, Pseudomonas, Hyphomonas, Sphingomonas, Arthrobacter, Planococcus, and Halobacillus. From this survey, it is proposed that ice diatom assemblages provide niches conducive to the proliferation of a diverse array of psychrophilic bacterial species.     

Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity

The extent to which cultured strains represent the genetic diversity of a population of microorganisms is poorly understood. Because they do not require culturing, metagenomic approaches have the potential to reveal the genetic diversity of the microbes actually present in an environment. From coastal California seawater, a complex and diverse environment, the marine cyanobacteria of the genus Synechococcus were enriched by flow cytometry-based sorting and the population metagenome was analysed with 454 sequencing technology. The sequence data were compared with model Synechococcus genomes, including those of two coastal strains, one isolated from the same and one from a very similar environment. The natural population metagenome had high sequence identity to most genes from the coastal model strains but diverged greatly from these genomes in multiple regions of atypical trinucleotide content that encoded diverse functions. These results can be explained by extensive horizontal gene transfer presumably with large differences in horizontally transferred genetic material between different strains. Some assembled contigs showed the presence of novel open reading frames not found in the model genomes, but these could not yet be unambiguously assigned to a Synechococcus clade. At least three distinct mobile DNA elements (plasmids) not found in model strain genomes were detected in the assembled contigs, suggesting for the first time their likely importance in marine cyanobacterial populations and possible role in horizontal gene transfer.     

Degradation of organic contaminants found in organic waste

In recent years, great interest has arisen in recycling of the waste created by modern society. A common way of recycling the organic fraction is amendment on farmland. However, these wastes may contain possible hazardous components in small amounts, which may prevent their use in farming. The objective of our study has been to develop biological methods by which selected organic xenobiotic compounds can be biotransformed by anaerobic or aerobic treatment. Screening tests assessed the capability of various inocula to degrade two phthalates di-n-butylphthalate, and di(2-ethylhexyl)phthalate, five polycyclic aromatic hydrocarbons, linear alkylbenzene sulfonates and three nonylphenol ethoxylates under aerobic and anaerobic conditions. Under aerobic conditions, by selecting the appropriate inoculum most of the selected xenobiotics could be degraded. Aerobic degradation of di(2-ethylhexyl)phthalate was only possible with leachate from a landfill as inoculum. Anaerobic degradation of some of the compounds was also detected. Leachate showed capability of degrading phthalates, and anaerobic sludge showed potential for degrading, polycyclic aromatic hydrocarbons, linear alkylbenzene sulfonates and nonyl phenol ethoxylates. The results are promising as they indicate that a great potential for biological degradation is present, though the inoculum containing the microorganisms capable of transforming the recalcitrant xenobiotics has to be chosen carefully.     

Bacteria which attack petroleum hydrocarbons in a saline medium

Bacterial strains were isolated from California coastal areas which showed the ability to oxidize normal paraffins, iso-paraffins, and aromatic hydrocarbons in a synthetic seawater medium. The ability to utilize a particular hydrocarbon was established not only on the basis of visible bacterial growth but also through a chromatographic technique which was standardized and which could define the amount of each hydrocarbon consumed by the bacteria in a mixture. Some of the strains exhibited vigorous hydrocarbon oxidation when exposed to synthetic mixtures of hydrocarbons as well as crude oil. Under conditions of aeration and agitation, mixed cultures could destroy approximately 50% of a South Louisiana crude oil in a period of 48 hr.     

Effects of Alkylbenzene Sulfonates on Yeasts

Species of Hansenula and Candida are resistant to high concentrations of anionic alkylbenzene sulfonates and degrade subinhibitory concentrations of these detergents.     

Methanogenic sarcina from an anaerobic microbial community degrading p-toluene sulfonate

The methanogenic strain MM isolated from an anaerobic microbial community degrading p-toluene sulfonate showed optimal values of temperature and pH for growth equal to 37 degrees C and 6.3-6.9, respectively. The doubling times of the isolate grown on methanol, acetate, and methylamines under the optimal conditions were 8.8, 19.1, and 10.3-28.1 h, respectively. The growth of strain MM was observed only when the cultivation medium contained casamino acids or p-toluene sulfonate. The G + C content of the DNA of the isolate was 40.3 mol%. This, together with DNA-DNA hybridization data, allowed the new isolate to be identified as a strain of the species Methanosarcina mazei. The new isolate differed from the known representatives of this species in that it was resistant to alkylbenzene sulfonates and able to demethylate p-toluene sulfonate when grown on acetate.     

PCR and Culture Identification of Pathogenic Leptospira spp. from Coastal Soil in Leyte,Philippines, after a Storm Surge during Super Typhoon Haiyan (Yolanda)

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthof''s medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.     

Distribution of Vibrio parahaemolyticus and Related Organisms in the Atlantic Ocean off South Carolina and Georgia

The distribution of Vibrio parahaemolyticus and related organisms in the Atlantic Ocean was determined during the summer of 1971 from samples collected at stations along four transects on the continental shelf off the South Carolina and Georgia coasts. No V. parahaemolyticus strains were isolated from any of the samples of seawater (surface and bottom), sediment, and plankton which were collected. A numerical taxonomy analysis of data on substrate utilization, including 154 organic compounds serving as single carbon sources, was carried out, and four groups of strains were observed. Each group showed well-separated distribution profiles from shore out to the continental shelf. That is, the groupings were observed to correspond to coastal, off-shore and intermediate distribution patterns for the strains. This study provides a useful example of the kind of ecological distributional analysis of bacteria which can be accomplished with numerical taxonomy.     

Two novel strains within the family Caulobacteraceae capable of degradation of linear alkylbenzene sulfonates as pure cultures

Culture-independent methods found members of the family Caulobacteraceae to be especially enriched in a soil microcosm perfused 21 days with a 50 mg l⁻¹ solution of linear alkylbenzene sulfonates (LAS) (Sánchez-Peinado et al., 2010). In this study, bacterial isolates from the same soil microcosm were screened for members of the family Caulobacteraceae. Two strains were identified as Phenylobacterium haematophilum and Caulobacter mirabilis according to their 16S rRNA gene sequences. The two strains were cultured in liquid medium containing LAS (20 and 40 mg l⁻¹), and HPLC analyses of the supernatants showed that they were capable of metabolizing LAS as pure cultures. These data confirm that indeed members of the family Caulobacteraceae were actively metabolizing LAS in the soil as previously reported, and provide with two valuable strains to study the metabolism of LAS in vitro.     

Habitat Segregation and Biochemical Activities of Marine Members of the Family Vibrionaceae

A comparative study of marine members of the family Vibrionaceae with the technique of numerical taxonomy revealed habitat segregation as well as a cosmopolitan nature of species distribution among the vibrios in different marine environments. The bacterial strains analyzed were isolated from seawater, sediments, phyto- and zooplankton, and fish in the Indian Ocean, the South and East China Sea, and West Pacific Ocean, and coastal areas of Japan. A total of 155 morphological, physiological, and biochemical tests were carried out for each of 405 strains examined. The results showed that most of the large taxonomical clusters which emerged from the computation corresponded to ecological groups which have particular niches. For instance, each group of seawater vibrios inhabited a particular water layer of limited depth range, in spite of the fact that strains of the group were isolated from sampling locations spread over a wide area from the Indian Ocean to Japanese coast. Various vibrio groups showed remarkable differences in their physiological and biochemical activities, and the activities of each group seemed to correspond with its ecological niche. The strains which inhabited surface-water layers grew fast and actively utilized many high-molecular-weight organic compounds and carbohydrates that are derived from fresh, easily degradable organic matter present in the surface waters, whereas the middle- and deep-water vibrios did not decompose most of the high-molecular-weight organic compounds except chitin but, rather, utilized some carbohydrates and organic acids which seemed to be derived from refractory particulate organic matter present in the deeper waters.     

Presence of pathogenic Vibrio Parahaemolyticus in waters and seafood from the Tunisian Sea

The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected from various exported seafood products comprising of fishes and shellfish (Mytilus edulis and Crassostrea gigas) or seawater, was studied. Eight strains were confirmed as V. parahaemolyticus by toxR -based polymerase chain reaction and only one strain out of these 8 strains was positive for tdh and trh genes. Toxigenic V. parahaemolyticus isolates are present in Tunisian coastal areas and they may also be present in Tunisian exported seafood products.     

Dynamics of seagrass bed microbial communities in artificial Chattonella blooms: A laboratory microcosm study

The influence of algicidal and growth-inhibiting bacteria in a seagrass (Zostera marina) bed, and their capability of controlling blooms of the fish-killing raphidophyte flagellate, Chattonella antiqua, were examined in laboratory microcosm experiments. Bacterial communities in seawater collected from the seagrass bed and Z. marina biofilm suppressed artificial Chattonella blooms in the presence of their natural competitors and predators. Phylogenetic analysis suggest that considerable numbers of bacteria that suppress Chattonella, including algicidal or growth-inhibiting bacteria isolated from seagrass biofilm and seawater from the seagrass bed, are members of Proteobacteria that can decompose lignocellulosic compounds. A direct comparison of partial 16S rRNA gene sequences (500 bp) revealed that the growth-limiting bacterium (strain ZM101) isolated from Z. marina biofilm belonged to the genus Phaeobacter (Alphaproteobacteria) showed 100% similarity with strains of growth-limiting bacteria isolated from seawater of both the seagrass bed and nearshore region, suggesting that the origin of these growth-limiting bacteria are the seagrass biofilm or seawater surrounding the seagrass bed. This study demonstrates that Chattonella growth-limiting bacteria living on seagrass biofilm and in the adjacent seawater can suppress Chattonella blooms, suggesting the possibility of Chattonella bloom prevention through restoration, protection, or introduction of seagrass in coastal areas.     

Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122.

Alcaligenes faecalis AE122 that used poly(3-hydroxybutyrate) (PHB) as a sole source of carbon was newly isolated from a coastal seawater sample. The strain required seawater for growth on PHB as well as in a nutrient broth, in which seawater could be replaced by an appropriate concentration of NaCl. PHB depolymerase was purified to homogeneity from the culture supernatant of A. faecalis AE122 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme consisted of a monomer subunit with a molecular mass of 95.5 kDa. The N-terminal amino acid sequence was GAWQNNLAGGFNKV. The dimeric and trimeric esters of 3-hydroxybutyrate were the main hydrolysis products of the purified enzyme. The enzyme was most active at pH 9.0 and 55 degrees C and was inhibited by phenylmethylsulfonyl fluoride. Several cations in seawater greatly enhanced the enzyme activity.     

Development of a heterotrophic bacterial community within a closed prawn aquaculture system

The quantitative and qualitative development of a heterotrophic bacterial community in seawater was studied throughout an experimental rearing of the prawnPenaeus japonicus. The maturation of juvenile prawns had been carried out for 8 months in aerated tanks of seawater without any water renewal. Bacteria (337 strains) were isolated from seawater, which had been sampled at different times. Samples from one of the mussels used to feed the prawns and from the digestive tract of one prawn each supplied 40 more strains. 101 tests were performed on each strain, and cluster analysis showed the existence of 4 different groups containing 95% of the sampled strains. Characteristics of the various samples and groups were described in terms of ecotype diversities, catabolic potentialities, nutritional capacities, and morpho-physiological groups. Until nitrification attained a steady state, the heterotrophic community clearly decreased in number and was composed mostly of auxotrophic bacteria (pseudomonads andMoraxella-Acinetobacter groups). These bacteria needed growth factors and were unable to use amino acids (group B). At the end of the experiment (7 months) the bacteria isolated from the water were vibrios and enterobacteria, as were those isolated from the prawn and the mussel. They clustered together in group A. The number of strains that clustered with those of natural seawater (group E) decreased steadily during the experimental period.