Enzymatic degradation of alginate by marine fungi

A total of 72 pre-selected strains of 19 species of marine fungi were tested for their ability to decompose sodium alginate, calcium alginate or freshly prepared calcium alginate gel. Active alginate decomposition was evident in 18 strains (25% of total tested). These belong to only three different species: Asteromyces cruciatus, Corollospora intermedia, and Dendryphiella salina. In broth culture, decomposition of sodium alginate by the two deuteromycetes was followed by gravimetric, electrometric, viscometric, photometric and chromatographic methods in order to characterize the alginase enzyme system and its degradation products. The alginase enzyme complex consisted of at least two different enzyme components: the already known alginate lyase (eliminase) and a new endo-alginate hydrolase. In summary, a model is presented on the alginase-mediated structural and molecular decomposition of sodium alginate by marine fungi.     

Ion Compartmentation in the Marine Fungus Dendryphiella salina in Response to Salinity: X-ray Microanalysis

X-ray microanalysis was performed on hyphae of the filamentousmarine fungus Dendryphiella salina growing at different salinitiesto give sodium, potassium and chloride concentrations in thecytoplasm, vacuole and cell wall. Sodium and chloride concentrationsincreased with salinity in all compartments. Cytoplasmic andvacuolar sodium and chloride concentration were broadly similar,and vacuolar contents represented, at most, 19% of the totalprotoplasmic content of an individual ion species. Potassiumconcentrations decreased to some extent with salinity, althoughconcentrations were not severely affected by competition withsodium uptake. Results are discussed with regard to the roleof ions in the overall osmotic adjustment in this species. Key words: Dendryphiella salina, marine fungus, salt-tolerance, x-ray microanalysis     

Efficiency of uronic acid uptake in marine alginate-degrading fungi

Despite the fact that many marine fungi, including phycomycetes, yeasts, ascomycetes and hyphomycetes, have been recorded from living and/or dead phaeophytes, only a few of these have been shown to be capable of degrading alginic acid or alginates. The degradation is achieved by the action of an exoenzyme complex, comprising alginate lyase, as well as alginate hydrolase activities. The latter was detected only recently by the authors. In this study, the growth of two marine sodiumalginate-degrading deuteromycetes,Asteromyces cruciatus andDendryphiella salina, was investigated, and the assimilation efficiency of sodiumalginate and its uronic acid degradation products, respectively, was estimated from the economic coefficient (E). E is calculated from the mycelial dry weight, divided by the weight of substrate consumed for this production. The economic coefficient forA. cruciatus was 48.6%, and that ofD. salina 38.9%. This indicates that the former species uses the alginate degradation products more efficiently than the latter. The observed E-values for the marine deuteromycetes agree with those from other fungi, e.g. terrestrial species. In general, it is concluded that the marine fungi appear to play a more important role in kelp-based ecosystems than was realized previously.     

Statistical Optimization of Culture Variables for Enhancing Agarase Production by <Emphasis Type="Italic">Dendryphiella arenaria</Emphasis> Utilizing <Emphasis Type="Italic">Palisada perforata</Emphasis> (Rhodophyta) and Enzymatic Saccharification of the Macroalgal Biomass

Agarase is a promising biocatalyst for several industrial applications. Agarase production was evaluated by the marine fungus Dendryphiella arenaria utilizing Palisada perforata as a basal substrate in semi-solid state fermentation. Seaweed biomass, glucose, and sucrose were the most significant parameters affecting agarase production, and their levels were further optimized using Box-Behnken design. The maximum agarase activity was 7.69 U/mL. Agarase showed a degree of thermostability with half-life of 99 min at 40 °C, and declining to 44.72 min at 80 °C. Thermodynamics suggested an important process of protein aggregation during thermal inactivation. Additionally, the enzymatic saccharification of the seaweed biomass using crude agarase was optimized with respect to biomass particle size, solid/liquid ratio, and enzyme loadings. The amount of biosugars obtained after optimization was 26.15 ± 1.43 mg/g. To the best of our knowledge, this is the first report on optimization of agarase in D. arenaria.     

Carbon source utilization by the marine Dendryphiella species D. arenaria and D. salina

Carbon utilization by the marine Dendryphiella species, D. arenaria and D. salina, was investigated to detect differences in utilization and traits associated with their adaptation to the marine habitat. Fifty-four strains were isolated world-wide and tested for the utilization of various carbon sources using BIOLOG phenotype MicroArray (PM) and for the production of extracellular enzymes on solid culture media and on API ZYM assay strips. PM analysis showed that the fastest growth occurred on several monosaccharides and amino acids, 2-keto-d-gluconic acid, succinamide and turanose. Some polyols were poor carbon sources. However, the two species differed in their utilization rates of carbon sources, forming three major clusters: two separate clusters for D. arenaria and D. salina and a third cluster in which strains of the two species formed separate subclades that correlated with geographic origin. Several carbon sources were also found useful in differentiating the two speices. Dendryphiella salina did not utilize xylitol and quinic acid, whereas D. arenaria grew well on these substrates. The latter failed to grow on sorbitol and grew slowly on mannitol, both were good substrates for the former. There were also no qualitative differences between the extracellular enzymes produced, although laccase and peroxidase activities were confined only to some strains. The physiological similarities exhibited by the two species support the close relationship between D. arenaria and D. salina.     

Electrophysiological Evidence for an Electrogenic Proton Pump and the Proton Symport of Glucose in the Marine Fungus Dendryphiella salina

The marine hyphomycete Dendryphiella salina (Suth.) Nicot &Pugh has a resting membrane potential of –250 mV (insidenegative). The respiratory inhibitors sodium azide and FCCPinduced a rapid but reversible depolarization of the membraneof at least 180 mV; sodium azide also caused alkalinizationof the medium. Vanadate brought about significant depolarizationbut this was not always reversible. EDTA induced depolarizationthough to a lesser extent. DIDS and SITS caused a depolarizationof around 30–70 mV which was readily reversible, N-ethylmaleimideirreversibly depolarized the membrane by 180–200 mV. Ouabainhad no effect. When external concentrations of H⁺ , K⁺ , Na⁺or Cl^– were changed singly, only changes in H⁺ affectedmembrane potential, with shifts decreasing with increasing pH.Glucose and 3-O-methyl glucose depolarized the membrane in aconcentration-dependent manner which was enhanced by starvationof the hyphae. Recovery occurred in the presence of the hexose.Glucose caused an alkalinization of the medium, with time characteristicssimilar to the membrane potential changes. It is concluded thatthere is an electrogenic proton pump and a proton—glucosesymporter in D. salina. The retention of proton-based transportsystems suggests a terrestrial origin for the fungus. Key words: Marine fungi, Dendryphiella salina, membrane potential, electrogenic proton pump, proton symport, hexose     

Molecular identification of β-carotene hyper-producing strains of Dunaliella from saline environments using species-specific oligonucleotides

Sequence-specific-oligonucleotides analysis has been used to identify Dunaliella bardawil, D. salina and D. parva from hypersaline environments based on their structural features of introns from the 18S rDNA. Carotenogenic and halophilic strains such as D. bardawil and D. salina were identified as harboring II and I introns within 18S rDNA, respectively. This is the first report on the existence of D. bardawil in saline water bodies of Mexico and Latin America.     

Antifouling activity of sessile bacilli derived from marine surfaces

Marine biofilms are a virtually untapped source of bioactive molecules that may find application as novel antifoulants in the marine paint industry. This study aimed at determining the potential of marine biofilm bacteria to produce novel biomolecules with potential application as natural antifoulants. Nine representative strains were isolated from a range of surfaces and were grown in YEB medium and harvested during the late exponential growth phase. Bacterial biomass and spent culture medium were extracted with ethanol and ethyl acetate, respectively. Extracts were assayed for their antifouling activity using two tests: (1) antimicrobial well diffusion test against a common fouling bacterium, Halomonas marina, and (2) anti-crustacean activity test using Artemia salina. Our results showed that none of the ethanolic extracts (bacterial biomass) were active in either test. In contrast, most of the organic extracts had antimicrobial activity (88%) and were toxic towards A. salina (67%). Sequencing of full 16 S ribosomal DNA analysis showed that the isolates were related to Bacillus mojavensis and Bacillus firmus. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) profiling of ethyl acetate extracts of culture supernatants showed that these species produce the bioactive lipopeptides surfactin A, mycosubtilin and bacillomycin D.     

Bioactivity in free-living and symbiotic cyanobacteria of the genus Nostoc

Fifty cyanobacterial strains from different habitats(symbioses, soil, fresh and marine waters) belongingto the genus Nostoc were cultured and tested forbioactivity. Thirty-seven strains were isolated in ourlaboratory, the remaining were supplied by officialculture collections. All the organisms were grownunder controlled laboratory conditions. The biomasseswere lyophilised and extracted with ethanol:water toobtain a hydrophilic extract and then withdichloromethane:isopropanol to obtain a lipophilicextract. Both crude extracts were tested forantifungal (against Penicillium expansum andRhizoctonia solani) and antibacterial activity(against Agrobacterium vitis, Escherichiacoli and Staphylococcus epidermidis), and fortoxicity against Artemia salina nauplii.Twenty-four strains showed activity against at leastone of the target organisms. Bioactivity was equallydistributed between lipophilic and hydrophilicextracts, and was mostly directed against fungi (15strains) and Artemia nauplii (12 strains);antibacterial activity was less frequent (8 strains).The presence of bioactivity was independent of thestrain origin.     

2株海洋来源产浅蓝霉素A的异壁放线菌的分离和鉴定

利用抗菌及卤虫致死活性模型,从中国南海海底沉积物来源的微生物中筛选到2株放线菌SCSIO WJ01和SCSIO ZJ63,其发酵产物具有较强活性,经16S rRNA基因序列分析这2株放线菌均为异壁放线菌Actinoalloteichus sp.。HPLC-DAD分析显示2株放线菌能产生同一个主要的次级代谢产物,通过正相硅胶柱色谱、反相中压柱色谱、半制备高效液相色谱等手段,从SCSIO WJ01的发酵产物中分离获得了该化合物,运用ESI-MS、1H及13C NMR波谱分析鉴定为浅蓝霉素A(Caerulomycin A)。此外,还从SCSIO WJ01的发酵产物中分离鉴定了浅蓝霉素D。     

DNA fingerprinting differentiation between β-carotene hyperproducer strains of Dunaliella from around the world

Background

Dunaliella salina is the most important species of the genus for β-carotene production. Several investigations have demonstrated that D. salina produces more than 10% dry weight of pigment and that the species grows in salt saturated lagoons. High plasticity in the green stage and the almost indistinguishable differences in the red phase make identification and differentiation of species and ecotypes very difficult and time consuming.

Results

In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and β-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico. All hyperproducer strains reached β-carotene levels of about 10 pg/cell. Optical microscopy did not allow to differentiate between these Dunaliella strains; however, 18S rDNA fingerprinting methodology allowed us to differentiate D. salina from D. salina/bardawil.

Conclusion

In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method. The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.     

Diet-induced variations in fatty acid content and composition of two on-grown stages of Artemia Salina

Three species of microalgae, the freshwater Euglena gracilis and themarine Dunaliella salina and Tetraselmis suecica, were fed tothe brine shrimp Artemia salina in order to compare their suitabilityin terms of fatty acid enrichment, and their effect on the biometric parametersof the zooplankter. The fatty acid content and composition were analyzed for the post-larval and pre-adult stages of Artemia fed the algae and theresults compared to the initial content of unfed 24-hour post-hatch nauplii.Differences in the total fatty acid content occurred between the three stages,the fatty acid profile being determined by the composition of the diet. A decreasing trend for almost all the individual fatty acids occurred throughdevelopment from post-larva to pre-adult with each of the three algal diets.Biometrical differences between Artemia fed the marine algae and that fed Euglena were not consistent in the post-larval stage, but became considerable in the pre-adult stage. Artemia fed with Euglena achieved twice the weight of animals fed the marine algae and showed thehighest length. The implications for the use of on-grown Artemia as afeed in larviculture of marine and freshwater fish and crustaceans are considered.     

PHYLOGENETIC RELATIONSHIP AMONG VARIOUS STRAINS OF DUNALIELLA (CHLOROPHYCEAE) BASED ON NUCLEAR ITS rDNA SEQUENCES

Phylogenetic analyses of 15 strains representing 8 taxa of Dunaliella (D. salina, D. bardawil, D. pseudosalina, D. tertiolecta, D. parva, D. viridis, D. peircei, and D. lateralis) belonging to both subgenera and all sections of the genus were carried out using the sequences of the nuclear rDNA spacers (internal transcribed spacer [ITS-1 + ITS-2]). The ITS data agreed with the traditional data in that D. lateralis (from subgenus Pascheria) is only distantly related to the seven taxa of the subgenus Dunaliella. The ITS data also supported the monophyly of the subgenus Dunaliella. Within the subgenus Dunaliella, sequence data resolved five phylogenetic groups; some isolates of D. parva and D. salina separated into different clades containing other species. For example, D. parva UTEX 1983 (section Dunaliella) grouped with D. viridis CONC 002 (section Virides); the former has more nucleotides in common with D. viridis (93.2% similarity) than to its conspecifics (85.6% similarity). Likewise, the strains of D. parva CCMP 362 and CCAP 19 / 9 (section Dunaliella), the three strains of D. tertiolecta (section Tertiolectae), and the one strain of D. peircei (section Peirceinae) formed a strong phylogenetic clade (99%–100% support). Dunaliella salina UTEX 200 is more closely related to D. pseudosalina CONC 010 than to its conspecifics (95% similarity), even though the two taxa differ markedly physiologically. The results revealed that D. parva UTEX 1983 has been misidentified and should be renamed as D. viridis. Similarly, the strains of D. parva CCAP 19 / 9 and CCMP 362 and the strain UTEX 2192 of D. peircei should be renamed as D. tertiolecta. More physiological and molecular work needs to be done to elucidate the correct taxonomic position of D. salina UTEX 200 and D. pseudosalina CONC 010. Finally, the high ITS sequence variability found among the various strains of D. salina underlines the importance of further work to elucidate the species status in this complex taxon.     

Bio-activity of marine cyanobacteria in the animal-based systems: Modulation of food intake, body weight and some haematological characters

With a view to assess the bioactive potential of the unexploited marine cyanobacteria available in abundance in the marine habitat, water-soluble fraction of the ethanolic extract of 12 different strains of marine cyanobacteria were administered to male rats and their impact on food intake, body weight and certain haematological characters including RBC counts, WBC-TC and DC, platelet counts, haemoglobin content and mean corpuscular haemoglobin was studied. The results suggest prospective applications as feed/nutritional supplements; among them, Spirulina subsalsa BDU 30311, Oscillatoria salina BDU 10142 and Phormidium valderianum BDU 20571 appear highly promising in this regard. Lyngbya sp. BDU 30601 and Pseudanabaena schmidlei BDU 30313 appear to contain one or more toxins and deter food intake and cause decrease in body weight. With Synechococcus elongatus BDU 30312 food intake increased over the control which was not commensurate with the change in body weight. The results relating to the haematological characters indicate bioactivity of the cyanobacteria in the animal-based systems, some of which are positive, whereas a few strains like Lyngbya BDU 30601 are negative, implying their pharmaceutical application.     

An aero-aquatic fungus, <Emphasis Type="Italic">Peyronelina glomerulata</Emphasis>, is shown to have teleomorphic affinities with cyphelloid basidiomycetes

On decayed wood near a stream, tiny cyphelloid, hair-bearing, Flagelloscypha-like basidiomata were found coexisting with conidia of an aero-aquatic fungus, Peyronelina glomerulata. An isolate originating from the basidioma produced conidia of P. glomerulata by soaking the culture in water. Three additional strains originating from conidia of P. glomerulata produced immature basidiomata with basidium-like structures on the agar medium after about 4 months incubation. Fine structure of the hyphal septa of P. glomerulata was found to be of the dolipore type. Phylogenetic analysis based on sequences from the D1/D2 regions of the LSU rDNA showed that the strains from conidia and from a basidioma clustered together in the Flagelloscypha clade and nested within the Nia clade of Hymenomycetes. The culture studies and molecular phylogenetic analysis suggested that P. glomerulata has a Flagelloscypha teleomorph, a cyphelloid basidiomycete. The molecular data also indicate that P. glomerulata is phylogenetically related to the marine basidiomycetes, Nia and Halocyphina. Thus, this study revealed that cyphelloid basidiomycetes have evolved into both marine as well as freshwater habitats by morphological adaptations of the teleomorphs in the former and of the anamorph in the latter case.     

Suitability of Selected Marine Algae for Growing the Marine Heterotrich Ciliate Fabrea salina1

ABSTRACT. Forty-five axenically grown algal (sensu lato) species representing six divisions—that is. 13 Chlorophyceae, 14 Chrysophycophyta, five Dinophycophyta, seven Cryptophycophyta, two Rhodophycophyta, and four Cyanochloronta—were aseplicaily presented separately as potential food sources to the marine helerotrich ciliate Fabrea salina under standardized algal number, medium, lighting, and temperature. The algae can be placed into three groups based on their effect on the intrinsic growth rate of the ciliate. Nutritious: Rhodomonas lens, cryptomonad LIS₁, Dunaliella parva, Prasinodadus marinus, Chroomonas salina, D. tertiolecta, Chaeloceros galvestonensis, D. primolecta, Phaeodactylum tricornutum, D. salina, Isochrysis galbana, Cylindrothecaclosterium, cryptomonad strains M₂, WH₂ & FSA, Chroomonas sp., P. lubricus, and Peridinium trochoideum. Maintamers: Cyanobacterium strain Tigriopus blue green, P. triquetum. Monochrysis lutheri, Exuviella gracilis, Platymonas tetrathele. Cyclotella caspa, Crypthecodinium cohnii, Prasinocladus C₅ strain, D. viridis, Nannochloris occulata, Tetraselmis gracilis, Anacystis marinum, Rhodosorus marinum, and Thalassiosira pseudonana. Nonnutritious: Stichococcus immobilis, Hymenomonas sp. strain 150, Syracosphaera sp. strain 181, Tetraselmis verrucosa, Thalassiosira fluviatilis, Microcoleus chthonoplastes, Synechococcus sp., Pavlova gyrans, Prymnesium parvum, Coccolithus huxleyi, Olisthodiscus luteus, Amphidinium carterii, and Porphyridium aerugineum. There was no apparent relationship between a given taxon and the nutritional value of the group, with the possible exception of the Cryptophycophyta.     

Purification and characteristics of fucoidanase obtained from <Emphasis Type="Italic">Dendryphiella arenaria</Emphasis> TM94

Dendryphiella arenaria TM94 is an obligate marine fungus. Fucoidanase expressed by TM94 by solid state fermentation was purified. The fermented solid medium was extracted with citric acid buffer, and the extracts were precipitated by acetone and separated on Sephadex G-100 chromatography. The specific fucoidanase activity of purified enzyme was 27-fold than that of the crude enzyme. The recovery of the enzyme was 17.69%. SDS-PAGE was used to identify the purity and the molecular weight of the fucoidanase. A single band appeared on SDS-PAGE gel which suggested that relatively pure fucoidanase has been obtained. The molecular weight of fucoidanase is 180 kDa and the isoelectric point was about pH 4.4. The purified fucoidanase appeared to have the maximum enzymatic activity at pH 6.0. K_M and the maximum velocity of the enzyme was 6.56 mg·mL⁻¹ and 6.55 mg·mL⁻¹·min⁻¹ by using fucoidan from Fucus vesiculosus as substrate. The enzyme may be a type of endo-fucoidanase which could hydrolyze high molecular weight fucoidan to low molecular weight fucoidan rather than to fucose.     

Screening for an oil‐removing microorganism and oil removal from waste silk by pure culture fermentation

The presence of oil is the major limitation to the regeneration of spun silk from waste silk. A pure culture fermentation process was developed to remove oil from waste silk. Fourteen strains were isolated from natural fermentation liquor of waste silk. The strain D3 showed highest lipase activity and oil‐removing ability. This strain was identified as Rhodococcus sp. on the basis of morphological and biochemical characteristics and 16S rRNA sequence analysis. The strain D3 was used to remove oil from waste silk by pure culture fermentation. The effects of various parameters on oil removal were investigated. A pH of 7.0, a temperature of 35–40°C, an incubation time of three days and an inoculum of 10% were optimum conditions for removing oil from waste silk by stain D3. This study shows that pure culture fermentation is a promising process to improve the oil removal of waste silk.     

有毒亚历山大藻对卤虫存活率和摄食率的影响

研究了有毒亚历山大藻对卤虫存活率和摄食率两方面的影响,得出以下结论：在卤虫存活率实验中,有毒亚历山大藻在2000cells／ml的密度下,对卤虫具有致死效应,卤虫在24-168h内全部死亡;在摄食实验中,有毒亚历山大藻对卤虫的摄食产生明显的抑制作用,卤虫对有毒藻的平均摄食率明显低于无毒藻组和混合实验组。在加入无毒藻东海原甲藻的混合培养状态下。卤虫存活率上升,30-60min摄食率增加,东海原甲藻在一定程度上可以减轻塔玛亚历山大藻对卤虫的毒害作用。有毒藻产生的PSP毒素并非导致卤虫死亡的主要原因,毒害作用可能与出现在卤虫体外的黏附物质有关。通过对3个不同生长期卤虫的研究发现,后无节幼体卤虫对有毒亚历山大藻的毒害作用最为敏感。