Screening for extracellular enzyme activities by bacteria isolated from samples collected in the Tyrrhenian Sea

Ninety-five bacterial isolates, obtained in pure cultures from samples collected in various sites of the East sector of the Tyrrhenian Sea, were plate-screened for their ability to produce twelve extracellular enzyme activities in order to find new strains for possible applications. Lipases, DNAase and RNAase were generally present; amilase, phosphatase, pectinase and protease were common. Chitinase and urease were present in a limited number of isolates while glucose oxidase and phenol oxidase were quite rare. Few isolates, producing a limited number of enzymes, could have a low eco-nutritional versatility while most of them, showing a diversified enzymatic competence, are probably more advantaged in the marine environment. However, none of the isolates was able to produce all the tested activities. Few strains (14) showed apparent high level of some extracellular enzyme activities and could be considered as potential high-producers.     

Properties of chitinolytic enzymes from the hepatopancreas of the red king crab (Paralithodes camtschaticus)

Our study confirms the presence of chitinolytic, chitosanolytic, and deacetylase activities in the hepatopancreas of the red king crab, related to the specific diet of this species. The maximum rate of chitin/chitosan hydrolysis by an enzyme preparation from crab hepatopancreas occurs at 36.5-37.0 degrees C. Two pH optimums have been found for the enzymatic reaction under mildly alkaline and acidic conditions for both exo- and endochitinase activities. The enzyme preparation is most affine to partly deacetylated chitin with an acetylation degree within 40-50%.     

Paenibacillus tylopili sp.nov., a chitinolytic bacterium isolated from the mycorhizosphere of Tylopilus felleus

Two chitinolytic bacterial strains (designated MK2^T and V7) were isolated from the mycorhizosphere of the fungus Tylopilus felleus. The strains were facultatively anaerobic G⁺ endospore formers. Physiological analysis and 16S rRNA gene PCR-RFLP assays revealed nearly identical profiles for both strains, demonstrating their relationship at the species level. Sequences specific for the genus Paenibacillus were found within the 16S rRNA gene sequence of the strain MK2^T. The 16S rRNA gene sequence showed the highest similarity to the sequences of Paenibacillus amylolyticus, P. pabuli and P. xylanilyticus. DNA-DNA relatedness of the strain with the type strain of P. amylolyticus was 4.95 %, of P. pabuli 38.0 %, and of P. xylanilyticus 46.3 %, indicating no relatedness between MK2^T and any of them at the species level. The most abundant fatty acids in strains MK2^T and V7 were anteiso-C_15:0, iso-C_16:0, iso-C_15:0 and n-C_16:0. DNA-DNA relatedness, morphological, physiological and chemotaxonomic analyses, and phylogenetic data based on 16S rRNA gene sequencing made it possible to describe both strains as the novel species of the genus Paenibacillus, for which the name Paenibacillus tylopili is proposed, the type strain being MK2^T (DSM 18927^T, LMG 23975^T).     

Carbohydrate synthesis by two Navicula strains isolated from benthic and pelagic mucilages in the Tyrrhenian Sea (Tuscan Archipelago)

Two exopolysaccharide (EPS)-producing strains of the diatom Navicula, were isolated from benthic and pelagic mucilaginous aggregates sampled in the Tyrrhenian Sea and cultured under laboratory conditions. The amount of carbohydrate formed over the growth period and on a per cell basis was quite similar. However, the benthic strain showed a preferential synthesis of the bound (i.e., cellular and capsular) carbohydrate fraction, whereas the pelagic strain preferentially synthesised soluble, polymeric carbohydrates. The polysaccharides released into the medium by the two strains showed the same qualitative monosaccharidic composition, being constituted by two acidic and six neutral sugars. It is suggested that the difference between the benthic and the pelagic strain in the synthesis of bound or soluble carbohydrates may be related to the different role of these compounds in the particular habitats of the strains.     

Analysis of demersal fish assemblages off the Southern Tyrrhenian Sea (central Mediterranean)

Monitoring and management of Mediterranean trawling requires a multispecies and ecosystem-based approach. Over the last 20 years studies on diversity and distribution of fish communities have been carried out by ecologists and applied to different ecosystems. Until now, very little information has been available on the Southern Tyrrhenian Sea. The aim of this work is: to analyse the structure of fish demersal assemblages, on a temporal (10 years) and spatial scale using a multivariate approach; to analyse, across space and time, the pattern of biodiversity by means of univariate indices; to identify areas supporting high values of diversity and evenness by a Geographical Information System (GIS) method. The analysis of 249 hauls from ten trawl surveys carried out between depths of 30 and 750 m in the South Tyrrhenian Sea yielded a total of 164 fish species. Multidimensional scaling ordination showed four groups distributed according to the depth gradient: a coastal group (0–100 m depth), a group in the lower part of the continental shelf (101–200 m), an epibathyal group (201–500 m) and the last one derived from hauls made in the middleslope (501–800 m). There were no differences, between years, protected and unprotected areas and geographical sectors. The whole study area was characterised by diversity values which were quite heterogeneous. The persistence index was generally low. Results could be useful from an ecological point of view and for the management of fishery activities.     

Occurrence of the scalloped ribbonfish Zu cristatus(Lampridiformes) in coastal waters of the central Tyrrhenian Sea, Italy

The occurrence of two individuals of Zu cristatus at 2 m depth in coastal waters of the Gulf of Castellamare (Tyrrhenian Sea, Italy) together with records of this rare pan‐Oceanic mesopelagic species is reported. Analyses of two mitochondrial genes (12 s and 16 s; 936 bp) revealed a 2·6% sequence divergence between Mediterranean and Pacific (Japanese) samples of the species.     

Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost

Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent K _m and the catalytic constant k_cat were 0.042 mg/ml and 7,588 min^?1, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way (“3?+?3”) but hydrolysis rate was much faster for (GlcN)₆ than (Glc)₆. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.     

Potentially toxic and harmful microalgae from coastal waters of the Campania region (Tyrrhenian Sea, Mediterranean Sea)

A total number of 40 potentially toxic microalgae and 5 taxa causing discolorations have been identified along the coasts of the Campania region (South Tyrrhenian Sea, Mediterranean Sea). This number is based on results of over 20 years of research at a coastal station in the Gulf of Naples, 4 years of monitoring activity along the Campania coasts and of a series of scattered and sporadic observations. Several species of the recently erected genera Karenia and Takayama are reported for the first time in the area. Information on the period of highest abundances or most probable period of occurrence indicates the late spring and summer as the periods of maximum risk of harmful events. Despite the variety of potentially toxic species, no human health problems nor fish kills have ever been recorded in Campania. The reasons for this apparent paradox are probably to be found in the ecological factors that regulate the abundance, toxicity and spatial distribution of the potentially harmful species and reflect as well the relatively low number of aquaculture plants in the area.     

Feeding ecology of Mediterranean razorfish Xyrichthys novacula in the Tyrrhenian Sea (Central Mediterranean Sea)

The stomach contents of 196 razorfish ( Xyrichthys novacula ), collected in the Tyrrhenian Sea (Ponza Island, Italy) between July and December 1994, were examined in order to assess the diet and to analyse size-related dietary variation of the species. The diet of X. novacula showed a strict relationship with well-sorted fine sand (SFBC) benthic species, mostly Mollusca and Echinodermata, as pointed out by the analysis at the specific level of the stomach contents. Mollusca Pelecipoda, essentially Acanthocardium tuberculatum and Echinodermata, with Echinocardium cordatum , made up 90% of the volume of prey of X. novacula. Mysidacea and Gastropoda were frequent but volumetrically less important. The study showed that male and female X. novacula were not effectively segregated by trophic dimension in the study area. The niche overlap between males and females in prey composition was pronounced, as evidenced by the significantly high values of Schoener's index with larger males that showed a greater tendency towards predation of large prey. The increment in length (L.I.) in males, despite a decrease in the percentage body weight increment (B.W.I.), could represent the result of a male reproductive feature: the lack of sperm competition allows the males much more energy to devote to growth than do that of the females of this species.     

Properties of chitinolytic enzymes from the hepatopancreas of the red king crab (<Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>)

Our study confirms the presence of chitinolytic, chitosanolytic, and deacetylase activities in the hepatopancreas of the red king crab, related to the specific diet of this species. The maximum rate of chitin/chitosan hydrolysis by an enzyme preparation from crab hepatopancreas occurs at 36.5–37.0°C. Two pH optimums have been found for the enzymatic reaction under mildly alkaline and acidic conditions for both exo-and endochitinase activities. The enzyme preparation is most affine to partly deacetylated chitin with an acetylation degree within 40–50%.     

太平洋克拉里昂-克利伯顿断裂带嘴刺目线虫多样性

从太平洋深海克拉里昂-克利伯顿断裂带(Clarion-Clipperton fracture zone,简称CC区)4个站位采集的深海沉积物样品中检出26条嘴刺目(Enoplida)线虫个体。综合应用形态学和分子生物学方法,共鉴定嘴刺目线虫6科8属,其中尖口线虫科(Oxystominidae)个体数量最多,占总数的57.7%,其次为前感线虫科(Anticomidae,19.2%)、光皮线虫科(Phanodermatidae,7.7%)、钩线虫科(Oncholaimidae,7.7%)、烙线虫科(Ironidae,3.8%)和矛线虫科(Enchelidiidae,3.8%)。科、属组成与相邻站点同期采样所获的线虫近似,而丰度组成比例有所差异。分子生物学方法获得了线虫rRNA基因序列16条,经Gen Bank数据库比对,其与已有的序列相似性范围为94%—99%,以此为依据可确定到科的水平和大部分属的水平(84.6%)。DNA条形码比对结果和形态学鉴定结果有较高一致性,表明分子条形码技术可作为深海线虫鉴定的有效手段。系统发育分析结果显示,基于18S和28S rRNA基因序列,采用不同方法构建系统发育树,其分支结构基本一致;钩线虫科和矛线虫科聚类在一起,光皮线虫科和前感线虫科聚类在一起,显示出彼此间较近的遗传关系。     

Biodeposition in a juvenile mussel bed of the east Frisian Wadden Sea (Southern North Sea)

Seasonal trends of biodeposition and animal coverage in backbarrier beds of the musselMytilus edulis in the East Frisian Wadden Sea were documented and quantified to assess the effects of physical disturbance by storms and to develop a conceptual model for the interpretation of biodeposits in the stratigraphic record. Accretion and erosion of biogenic muds (faeces and pseudofaeces) were quantified by repeated precision levelling of a 12 m² test area. Animal coverage was documented by vertical photography prior to each levelling survey. Levelling and photography were conducted from a bridge and rail system to avoid physical disturbance by trampling. Four surveys, covering a period of different weather conditions, showed variable mean accretion/erosion rates of biodeposits ranging from +0.56 mm d⁻¹ (accretion) to −1.34 mm d⁻¹ (erosion) and areal coverage of mussels ranging from 39.1% to 0%. A pronounced seasonal trend in the biodepositional pattern was observed. Mean accretion rates were high (>0.5 mm d⁻¹) during the summer season, whereas in the winter season net deposition decreased to zero or showed erosional trends. Longterm sediment budgets are thus composed of highly variable mean annual rates which, in turn, comprise highly variable seasonal and even monthly rates, depending on the local weather pattern. This forms the basis for a conceptual model, in terms of which the stratigraphic record of fossil biodeposits can be meaningfully interpreted.     

First record of microplastics ingestion by European hake MERLUCCIUS MERLUCCIUS from the Tyrrhenian Sicilian coast (Central Mediterranean Sea)

A sample of 67 European hake Merluccius merluccius were examined to highlight the ingestion of microplastics in the Tyrrhenian Sea. In all samples, 31 black fibres were found in the stomach contents corresponding to 46.3% of the specimens. The data presented here could be important for the implementation of the EU Marine Strategy Framework Directive in Mediterranean waters.     

First record of a lecithotrophic nauplius in Harpacticoida (Crustacea,Copepoda) collected from the Weddell Sea (Antarctica)

Summary During an investigation of the meiofauna communities of the eastern Weddell Sea (Antarctica) the first naupliar stage of a new species of Pseudotachidius T. Scott, 1897 (Thalestridae) was held in culture on board R V Polarstern. These nauplii are extraordinary for a harpacticoid copepod in being globular in shape and densely filled with yolk. They lack mouth, gut tube, anus and the masticatory parts of the appendages, all of which indicate that they do not feed. It is not unlikely that later stages are also able to subsist safely on the large yolk reserves.     

Microbial Diversity at a Hot,Shallow-Sea Hydrothermal Vent in the Southern Tyrrhenian Sea (Italy)

Microbial community thriving at shallow hydrothermal vent named Black Point, close to the Island of Panarea in the Southern Tyrrhenian Sea (Italy), was studied by microscopic, cultural and molecular methods. New strains of Bacillus and Geobacillus were isolated. Members of sulphur-oxidizing bacteria, belonging to the genera Halothiobacillus and Thiomicrospira were demonstrated by both culture-dependent and -independent methods. Genetic diversity of Bacteria was higher than that of Archaea. Bacterial 16S rRNA gene sequences from Black Point showed low levels of similarity with other yet deposited sequences, suggesting that new bacterial phylotypes are present in this site. Dominant populations of Bacteria belonged to uncultured members of Proteobacteria (mainly of the class Gammaproteobacteria), Firmicutes and Acidobacteriaceae. Primary production appeared to be supported by chemosynthetic and photosynthetic bacteria affiliated with members of Gammaproteobacteria. Most of the archaeal sequences matched those of yet-uncultivated Archaea, thus their role at this vent is not well understood.     

Complete mineralisation of dimethylformamide by Ochrobactrum sp. DGVK1 isolated from the soil samples collected from the coalmine leftovers

A bacterial strain DGVK1 capable of using N,N-dimethylformamide (DMF) as sole source of carbon and nitrogen was isolated from the soil samples collected from the coalmine leftovers. The molecular phylogram generated using the complete sequence of 16S rDNA of the strain DGVK1 showed close links to the bacteria grouped under Brucellaceae family that belongs to alphaproteobacteria class. Specifically, the 16S rDNA sequence of strain DGVK1 has shown 97% similarity to Ochrobactrum anthropi LMG 3331 (D12794). This bacterium has also shown impressive growth on dimethylamine, methylamine, formaldehyde and formate that are considered to be the prominent catabolic intermediates of DMF. DMF degradation has led to the accumulation of ammonia and dimethylamine contributing to the increase of pH of the medium. The DMF-grown resting cells of Ochrobactrum sp. DGVK1 have also contributed for the release of ammonia when resting cell suspension was added to phosphate buffer containing DMF. Similar experiments done with the glucose-grown cultures have not produced ammonia and thus indicating the inducible nature of DMF-degrading enzymes in Ochrobactrum sp. DGVK1. Further, dimethylformamidase, dimethylamine dehydrogenase and methylamine dehydrogenase, the key enzymes involved in the degradation of DMF, were assayed, and the activities of these enzymes were found only in DMF-grown cultures further confirming the inducible nature of the DMF degradation. Based on these results, DMF degradation pathway found in Ochrobactrum sp. DGVK1 has been proposed.     

Age,growth and feeding habits of the bluemouth rockfish,Helicolenus dactylopterus dactylopterus (Delaroche 1809) in the central Mediterranean (southern Tyrrhenian Sea)

This paper provides data on age, growth and diet of the bluemouth rockfish, Helicolenus dactylopterus dactylopterus (Scorpaenidae), central Mediterranean Sea. Estimated by otolith readings, ages ranged from 0 to 21 years. Parameters of the von Bertalanffy growth curves were L_inf = 26.1 cm, k = 0.14 year^?1, t₀ = ?1.92 years and the growth performance index was Φ = 1.98. The weight‐length relationship, W = 0.016 TL^2.99; R² = 0.99, described an isometric growth of the species. This species is a carnivore focusing mainly on the crustaceans Goneplax rhomboides and Lophogaster typicus. Statistical analysis showed an ontogenetic shift between small (4.0–6.3 cm) and larger fishes. Significant differences were also found in relation to the sampling depth.     

Fe(III) oxide reduction and carbon tetrachloride dechlorination by a newly isolated Klebsiella pneumoniae strain L17

Aims:  To isolate an iron-reducing bacterium and examine its ability of Fe(III) oxide reduction and dechlorination.
Methods and Results:  A fermentative facultative anaerobe, strain L17 isolated from subterranean sediment, can reduce Fe(III) oxides and carbon tetrachloride (CT). It was identified as Klebsiella pneumoniae by 16S rRNA sequence analysis. Strain L17 can metabolize fermentable substrates such as citrate, glycerol, glucose and sucrose coupled with the reduction of hydrous ferric oxide, goethite, lepidocrocite and hematite. Fe(III) reduction was influenced by crystal structure of Fe(III) oxide, type of fermentable substrate, metabolic status of the strain, and significantly enhanced by addition of anthraquinone-2,6-disulfonate (AQDS). Strain L17 could dechlorinate CT to chloroform, and the rate was accelerated in the presence of Fe(III) oxide and AQDS. Biotic dechlorination by strain L17 and abiotic dechlorination by sorbed Fe(II) were proposed as the two main mechanisms. AQDS might accelerate the dechlorination by transferring electrons from strain L17 to Fe(III) oxide and CT.
Conclusions:  K. pneumoniae L17 can reduce Fe(III) oxides and CT. The two reductions can occur simultaneously, and be significantly promoted by AQDS.
Significance and Impact of the Study:  This is the first report of a strain of K. pneumoniae capable of reducing Fe(III) oxides and CT. As a strain of environmental origin, strain L17 may have the potential for bioremediation of chlorinated compound-contaminated sites.     

Characterization of a sialidase (neuraminidase) isolated from Clostridium chauvoei (Jakari strain)

A sialidase from Clostridium chauvoei (Jakari strain), an indigenous bacterial strain that causes blackleg in Nigerian cattle and other ruminants was isolated and partially purified by chromatography on DEAE cellulose, hydroxyapatite and phenyl agarose columns. The enzyme migrated as a 65-kDa protein after electrophoresis on sodium dodecyl sulphate polyacrylamide gels. It was optimally active at pH 4.5 and 40 degrees C with an activation energy (Ea) of 13.40 kJ mol(-1). It had Km and Vmax values of 170 microM and 200 micromole h(-1) mg(-1) respectively with fetuin as substrate. When sialyllactose (Neu5Ac2,3 lactose) was used as substrate the Km and Vmax values were 8 microM and 5 micromoles min(-1) mg(-1) respectively. The Clostridium chauvoei sialidase cleaved sialic acids from RBC ghosts of sheep, horse, goat, cattle, pig and mice as well as mouse brain cells, albeit at different rates. The enzyme was activated by Ca2+ and Mg2+ and inhibited by the group-specific reagents diethylpyrocarbonate (DEP) and N-ethylmalemide (NEM). The sialidase inhibitors, 2,3 didehydroneuraminic acid (Neu5Ac2,3en) and paranitrophenyl oxamic acid (pNPO) inhibited the enzyme competitively with Ki values of 40 and 30 microM respectively.