Screening Methods for Improving Rotifer Culture Quality

We studied whether the selection of rotifer B. plicatilis strain (Japanese, Russian or Australian), as well as the addition of gamma-aminobutyric acid (GABA) to the culture water, are useful in stabilising rotifer cultures. We examined the effect of a combination of the following stressors: unionized ammonia (2.4 mg l⁻¹), contamination by protozoa Euplotes sp. (10 cells ml⁻¹) and addition of methyl cellulose to increase the culture water viscosity at 15 cp. Rotifer reproductive tests and enzyme activity measurements (glucosidase) were conducted to determine the effect of the treatments. All tests were conducted at 25 °C and rotifers were fed Nannochloropsis oculata at 7 × 10⁶ cells ml⁻¹. The combined effects of the stressors caused a significant decrease in lifespan, fecundity and glucosidase activity. The effect of the stressors on reproductive characteristics and glucosidase activity could be neutralized if rotifers were treated with GABA.     

Effect of Abiotic Stress on Phosphate Solubilization by Biocontrol Fungus <Emphasis Type="Italic">Trichoderma</Emphasis> sp.

This study was undertaken to explore the role of Trichoderma sp. in phosphate (P) solubilization and antagonism against fungal phytopathogens. All fungal isolates (SE₆, KT₆, KT₂₈, and BRT₁₁) and a standard culture of T. harzianum (Th-std) were able to antagonize two fungal phytopathogens (Sclerotium rolfsii and Rhizoctonia solani) of chickpea (Cicer arietinum L.) wilt complex. Transmission electron microscopic studies (TEM) further confirmed ultra-cytological changes in the sclerotia of S. rolfsii parasitized by Trichoderma sp. All fungal cultures exhibited production of NH₃ and siderophore, but only BRT₁₁, SE₆, and Th-std could produce HCN. Among all the cultures tested, isolate KT₆ was found to be most effective for solubilization of ferric phosphate releasing 398.4 μg ml⁻¹ phosphate while isolates BRT₁₁ and SE₆ showed more potential for tricalcium phosphate (TCP) solubilization releasing 449.05 and 412.64 μg ml⁻¹ phosphate, respectively, in their culture filtrates. Part of this study focused on the influence of abiotic stress conditions such as pH, temperature, and heavy metal (cadmium) on phosphate (TCP) solubilizing efficiency. Two selected cultures KT₆ and T. harzianum retained their P solubilizing potential at varying concentrations of cadmium (0–1000 μg ml⁻¹). Isolate KT₆ and standard culture of T. harzianum released 278.4 and 287.6 μg ml⁻¹ phosphate, respectively, at 1000 μg ml⁻¹cadmium. Maximum solubilization of TCP was obtained at alkaline pH and at 28°C temperature. Isolate BRT₁₁ was found most alkalo-tolerant releasing 448.0 μg ml⁻¹ phosphate at pH 9.     

The effect of <Emphasis Type="Italic">Poterioochromonas</Emphasis> abundance on production of intra- and extracellular microcystin-LR concentration

Due to its capability for producing various microcystins, Microcystis aeruginosa is recognized as one of the most toxic, bloom-forming cyanobacteria. In this study, the fates of intra- and extracellular microcystin-LR (MC-LR) were investigated when the mixotrophic golden alga Poterioochromonas sp. (ZX1) was grazing on M. aeruginosa cells. In the control groups, the total MC-LR concentration increased with the growth of M. aeruginosa with an MC-LR content per cell of 0.5–1.5 × 10⁻⁸ μg cell⁻¹. In the treatment with ZX1, the total MC-LR decreased linearly throughout the incubation period. In particular, intracellular MC-LR disappeared with a loss of M. aeruginosa cells in the first few days. Part of the intracellular MC-LR was released to the medium under the grazing stress, resulting in an increase of extracellular MC-LR. The degradation rate of MC-LR was positively related to the initial abundance of ZX1 and negatively related to that of M. aeruginosa. The inhibition ratio of MC-LR production dropped sharply from 98 to 67% when the initial abundance of M. aeruginosa increased from 10⁶ to 10⁷ cells ml⁻¹. However, it increased from 84 to 99% when the initial ZX1 abundance increased from 10⁴ to 10⁵ cells ml⁻¹. The effective removal of both M. aeruginosa cells and MC-LR was observed under lower M. aeruginosa abundance (<10⁶ cells ml⁻¹) and higher ZX1 abundance (>1% of M. aeruginosa abundance). Light had little impact on MC-LR degradation, but MC-LR degradation decreased due to the loss of ZX1 after 10 days of darkness. This study showed that the interactions between M. aeruginosa and ZX1 were strongly influenced by their initial abundances.     

Antimicrobial activity of an endophytic <Emphasis Type="Italic">Xylaria</Emphasis> sp.YX-28 and identification of its antimicrobial compound 7-amino-4-methylcoumarin

An endophytic Xylaria sp., having broad antimicrobial activity, was isolated and characterized from Ginkgo biloba L. From the culture extracts of this fungus, a bioactive compound P3 was isolated by bioactivity-guided fractionation and identified as 7-amino-4-methylcoumarin by nuclear magnetic resonance, infrared, and mass spectrometry spectral data. The compound showed strong antibacterial and antifungal activities in vitro against Staphylococcus aureus [minimal inhibitory concentrations (MIC) 16 μg·ml⁻¹], Escherichia coli (MIC, 10 μg·ml⁻¹), Salmonella typhia (MIC, 20 μg·ml⁻¹), Salmonella typhimurium (MIC, 15 μg·ml⁻¹), Salmonella enteritidis (MIC, 8.5 μg·ml⁻¹), Aeromonas hydrophila (MIC, 4 μg·ml⁻¹), Yersinia sp. (MIC, 12.5 μg·ml⁻¹), Vibrio anguillarum (MIC, 25 μg·ml⁻¹), Shigella sp. (MIC, 6.3 μg·ml⁻¹), Vibrio parahaemolyticus (MIC, 12.5 μg·ml⁻¹), Candida albicans (MIC, 15 μg·ml⁻¹), Penicillium expansum (MIC, 40 μg·ml⁻¹), and Aspergillus niger (MIC, 25 μg·ml⁻¹). This is the first report of 7-amino-4-methylcoumarin in fungus and of the antimicrobial activity of this metabolite. The obtained results provide promising baseline information for the potential use of this unusual endophytic fungus and its components in the control of food spoilage and food-borne diseases.     

Antarctic sea ice viral dynamics over an annual cycle

Viral abundance, burst sizes, lytic production and temperate phage were investigated in land-fast ice at two sites in Prydz Bay Antarctica (68°S, 77°E) between April and November 2008. Both ice cores and brine were collected. There was no seasonal pattern in viral or bacterial numbers. Across the two sites virus abundances ranged between 0.5 × 10⁵ and 5.1 × 10⁵ viruses ml⁻¹ in melted ice cores and 0.6 × 10⁵–3.5 × 10⁵ viruses ml⁻¹ in brine, and bacterial abundances between 2.7 × 10⁴ and 17.3 × 10⁴ cells ml⁻¹ in melted ice cores and 3.9 × 10⁴–32.5 × 10⁴ cells ml⁻¹ in brine. Virus to bacterium ratios (VBR) showed a clear seasonal pattern in ice cores with lowest values in winter (range 1.2–20.8), while VBRs in brine were lower (0.2–4.9). Lytic viral production range from undetectable to 2.0 × 10⁴ viruses ml⁻¹ h⁻¹ in ice cores with maximum rates in September and November. In brine maximum, lytic viral production occurred in November (1.18 × 10⁴ viruses ml⁻¹ h⁻¹). Low burst sizes were typical (3.94–4.03 viruses per bacterium in ice cores and 3.16–4.0 viruses per bacterium in brine) with unusually high levels of visibly infected cells—range 40–50%. This long-term investigation revealed that viral activity was apparent within the sea ice throughout its annual cycle. The findings are discussed within the context of limited data available on viruses in sea ice.     

Antimicrobial activity of crude extracts from mangrove fungal endophytes

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml⁻¹). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml⁻¹). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml⁻¹ and 8–200/8–200 μg ml⁻¹, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml⁻¹). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml⁻¹against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products.     

Production of cyclodextrin glucanotransferase from an alkalophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. by pH-stat fed-batch fermentation

Batch and fed-batch fermentation processes were employed to culture an alkalophilic Bacillus sp. for the production of cyclodextrin glucanotransferase (CGTase). CGTase production was repressed by glucose and induced by soluble starch. By fed-batch fermentation, a CGTase activity up to 56 unit ml⁻¹ with 65 g dry cells l⁻¹ were achieved. The CGTase activity and cell density were increased 360 and 510%, respectively, from those values achieved with batch fermentation.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

High density cultivation of Dictyostelium discoideum in a rotating polyurethane foam-bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In order to achieve high cell density cultivation, polyurethane foam (PUF) with high porosity was introduced as new matrix for the immobilization of D. discoideum. The results showed that about 88–93% cells of D. discoideum were adsorbed onto the PUF particles after 100 min equilibrium between adsorbed and free cells, and the highest immobilization rate was achieved by adding the same quantity of PUF matrix with the thin cylinder style. Furthermore, polyurethane foam was used as the immobilization matrix in a rotating PUF-bed bioreactor system. With batch cultures in the rotating bed bioreactor, the concentration of immobilized cells in the PUF carrier increased to 4.2 × 10⁷ cells ml⁻¹ after 167 h cultivation, which was about fourfold higher than the maximal cell density in the conventional free-cell culture. Further studies showed that the cells of D. discoideum were not just simply adsorbed on the surfaces, but actively attached to the surfaces through their network of pseudopodia or filopodia. The present work is very promising to improve the productivity of recombinant proteins in D. discoideum with high cell density in this novel rotating bed bioreactor.     

Automatic detection of Staphylococcus aureus and Shigella dysenteriae with separated electrodes series piezoelectric sensing technique

The series piezoelectric quartz crystal (SPQC) sensing technique is a rapid and sensitive method for detection of microorganisms. In the present study, the detection device was composed of detecting system, signal generating system and data analyzing system. To magnify the amount of detection samples, eight independent SPQC sensors were parallel connected to form a muti-channel detecting unit. Electrodes were separated from the SPQC sensor and immersed into culture medium to detect the change of solution conductivity. The cell constant k was determined as 0.05 m, and the sensitivity interval of the device was from 550 to 600 μs. To maintain sensitivity of the SPQC sensor, a novel culture medium amino acid broth (AaB) was developed. It was nutrient with low conductivity and satisfied our detection device. For determining frequency detection time (FDT) expediently and accurately, FDT was defined afresh with fitting–differentiating method. Pathogens Staphylococcus aureus and Shigella dysenteriae were determined with an automated detecting device and the methods mentioned above. The calibration curves of FDT against density of bacteria showed a linear correlation coefficient (R ≥ 0.99) over the range of 10–10⁶ cells ml⁻¹. Detection results all fell inside the 95% confidence interval of a standard pour plate counting method. The reproducibility was also reviewed, and results showed that the device was stable and sensitive even after 180 days of employment.     

Stimulation of menthol production in <Emphasis Type="Italic">Mentha piperita</Emphasis> cell culture

Plant cell culture provides an alternative means for producing secondary metabolites. In this study, experiments were carried out to study the impact of several parameters, independently and in combination, on the stimulation of menthol production in the cell suspension culture of Mentha piperita. Callus was obtained from leaf segments of in vitro grown plantlets on Murashige and Skoog (MS) medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid to initiate cell suspension culture. This culture was maintained in half-strength MS medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid at 15 d interval and used for further studies. Precursor feeding alone, i.e., menthone, at 35 μM concentration showed slightly improved productivity. γ-Cyclodextrin alone at 60 μM concentration and in combination with menthone feeding at 35 μM increased menthol yield up to 92 and 110 mg l⁻¹ in comparison to 77 mg l⁻¹ of control culture. Synergistic potentiation effect of menthone feeding at 35 μM and γ-cyclodextrin at 60 μM treatment followed by in situ adsorption with RP-8 also showed potential stimulation of menthol production in M. piperita cell culture. Fungal elicitor treatment showed enhanced production level up to 140.8 mg l⁻¹ in comparison to that of control. Further studies were carried out with the establishment of Agrobacterium tumefaciens (Ach5) gall-mediated calli, and consequently, cell suspension culture and results showed the significant enhancement of menthol yield up to 278 mg l⁻¹. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A comparison of phytoplankton size-fractions in Mondsee,an alpine lake in Austria: distribution,pigment composition and primary production rates

Production rates, abundance, chlorophyll a (Chl a) concentrations and pigment composition were measured for three size classes (<2 μm, 2–11 μm and >11 μm) of phytoplankton from May to December 2000 in deep, mesotrophic, alpine lake Mondsee in Austria. The study focuses on differences among phytoplankton size fractions characterised by their surface area to volume ratio ([mm² l⁻¹: mm³l⁻¹]), pigment distribution patterns and photosynthetic rates. Particular attention was paid to autotrophic picophytoplankton (APP, fraction <2 μm) since this size fraction differed significantly from the two larger size fractions. Among the three fractions, APP showed the highest surface area to volume ratios and a high persistence in the pattern of lipophilic pigments between temporarily and spatially successive samples (about 80% similarity of pigment composition between samples over seasons and depths). The epilimnetic abundance of APP varied seasonally with an annual maximum of 180 × 10³ cells ml⁻¹ in June (at 4–9 m). The minimum (October at 12 m) was more than an order of magnitude lower (4.9 × 10³ ml⁻¹). APP peaked during autumn and contributed between 24% and 42% to the total area-integrated Chl a (10–23 mg m⁻²) and between 16% and 58% to total area-integrated production (5–64 mg m⁻²  h⁻¹) throughout seasons.     

Degradation of chloroform by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in calcium alginate beads

A Bacillus sp., capable of degrading chloroform, was immobilized in calcium alginate. The beads in 20 g alginate l⁻¹ (about 2 × 10⁸ cells/bead) could be re-used nine times for degradation of chloroform at 40 μM. The immobilized cells had a higher range of tolerance (pH 6.5–9 and 20–41°C) than free cells (pH 7–8.5 and 28–32°C). At 5 g alginate l⁻¹, leakage of the cells from the beads was 0.51 mg dry wt ml⁻¹. This species is the first reported Bacillus that can degrade chloroform as the sole carbon source.     

Callus formation,plant regeneration,and transient expression in the halophyte sea aster (<Emphasis Type="Italic">Aster tripolium</Emphasis> L.)

An in vitro regeneration and transient expression systems were developed for the halophyte sea aster (Aster tripolium L.), an important genetic resource for salt tolerance. Adventitious shoots were formed from both leaf explants and suspension-cultured cells in a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) basal salts containing 500 mg l⁻¹ casamino acids, and supplemented with 5.4 μM a-naphthaleneacetic acid (NAA) and 4.7 μM kinetin to the culture medium. Hyperhydricity of shoots was avoided by increasing the ventilation of the culture vessel. Root formation from shoots was promoted in the presence of 26.9 μM NAA. A high yield of protoplasts was isolated using 1% cellulase and 0.25% pectinase from both leaf mesophyll and suspension-cultured cells, and these were used for transient expression. The highest level of transient expression of the green fluorescent protein was obtained with 1 × 10⁵ protoplasts ml⁻¹, 25 μg batch⁻¹ of plasmid vector, and 30% polyethylene glycol 4,000.     

Regulation of inorganic carbon acquisition by nitrogen and phosphorus levels in the Nannochloropsis sp.

Nannochloropsis sp. was grown to the exponential phase and transferred to the high CO₂ (2,800 μl l⁻¹) and irradiance (100 μmol photons m⁻² s⁻¹) condition with different levels of nitrate and phosphate for 72 h, then the photosynthetic activity and inorganic carbon acquisition of the alga were measured. The apparent photosynthetic efficiency (α) of Nannochloropsis sp. decreased with increasing NO₃ ⁻ concentration from 150 to 3,000 μM, and the high nitrate-grown cells showed the lowest levels of light-saturated photosynthetic rate (P _m), while the low nitrate-grown cells showed the highest levels of dark respiration rate (R _d). The maximal light-saturated photosynthetic rate and the minimal dark respiration rate were seen under the middle nitrate condition. When the nitrate concentration ranged from 150 to 3,000 μM, the affinity for inorganic carbons of Nannochloropsis sp. increased sharply with the increasing NO₃ ⁻ concentration to 300 μM and then decreased significantly. The middle phosphate-grown cells exhibited the highest light-saturated photosynthetic rate and apparent photosynthetic efficiency, however, the affinity for inorganic carbons of Nannochloropsis sp. was the maximum under the low phosphate condition. It was shown that the appropriate nitrogen and phosphorus levels were of vital importance to the photosynthesis of cells.     

Identification and control of bacterial contaminants from Ilex dumosa nodal segments culture in a temporal immersion bioreactor system using 16S rDNA analysis

Endogenous bacterial contaminants isolated from infected cultures of Ilex dumosa nodal segments were identified as Stenotrophomonas maltophilia and Achromobacter sp. using 16S rDNA analysis. A range of antibiotics with different mechanism of actions and the commercial biocide PPM™ were tested for their capacity to repress the growth of Gram negative bacteria grown in liquid medium during the establishment phase of temporal immersion systems. The best results were obtained with the addition of 0.5 mg ml⁻¹ cefotaxime to the culture media obtaining 100% of uncontaminated cultures without suppress of shoot growth.     

Efficient protocol for in vitro production of androgenic haploids of <Emphasis Type="Italic">Phlox drummondii</Emphasis>

Production of haploid plants has been restricted to only a few ornamental species. In this paper an efficient anther culture protocol has been devised for production of haploid plants of Phlox drummondii, a garden ornamental. Anthers with microspores at early- to late-uninucleate stages were inoculated on MS (Murashige and Skoog, Physiol Plant 15:473–479, 1962) basal medium containing 9% sucrose, 10 μM 2,4-D + 5 μM BA in the dark for callus induction. The callus (~2 mm) was transferred to MS medium containing 3% sucrose + 10 μM BA + 5 μM NAA under a 16 h photoperiod for multiplication. Anther-derived callus showed the greatest shoot differentiation (60% with greater than 3 shoots per culture) at 13 weeks after culture initiation when maintained on MS medium supplemented with 3% sucrose and cytokinin (7.5 μM BA). At 68 weeks, only 4.6% of cultures differentiated with less than one shoot per callus. Anther-derived shoots rooted readily on MS medium containing 7.5 μM IAA. Of 60 plants that regenerated from anther callus, 50% were haploid, 30% diploid, and 20% aneuploid. Developed protocol could be useful for the haploid induction of outcrossing ornamental plants for production of their homozygous double haploids.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Gene cloning,expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium <Emphasis Type="Italic">Agarivorans</Emphasis> sp. HZ105

A β-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity, agarase HZ2 was assigned to the glycoside hydrolase family 50. The β-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized β-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization above four, to yield neoagarotetraose as the dominant product, which was different from β-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type β-agarase. Beta-mercaptoethanol (90 mM) and dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the activity completely. The agarase activity was independent of Na⁺, K⁺, Mg²⁺ and Ca²⁺. The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m values toward agarose of agarase rHZ2 were 5.9 mg ml⁻¹, 235 U mg⁻¹, 401 s⁻¹ and 6.8 × 10⁵ M⁻¹ s⁻¹, respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.     

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III under laboratory culture

Interactions between Prorocentrum donghaiense Lu and Scrippsiella trochoidea (Stein) Loeblich III, two species of causative bloom dinoflagellates in China, were investigated using bi-algal cultures under controlled laboratory conditions. The growth of P. donghaiense and S. trochoidea were significantly suppressed when the initial cell densities were set at 1.9 × 10⁴ cells mL⁻¹ or 1.9 × 10⁵ cells mL⁻¹ for P. donghaiense and 1.0 × 10⁴ cells mL⁻¹ for S. trochoidea when the initial size/density ratio was 1:1 or 10:1, respectively, but no out-competement was observed in either bi-algal culture by the end. The simultaneous assay on the culture filtrate showed that P. donghaiense filtrate prepared at a lower initial density (1.9 × 10⁴ cells mL⁻¹) stimulated the co-cultured S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹, but filtrate at a higher density (1.9 × 10⁵ cells mL⁻¹) depressed its growth. Differently, the filtrate of S. trochoidea at a density of 1.0 × 10⁴ cells mL⁻¹ significantly suppressed the growth of P. donghaiense at a density of 1.9 × 10⁴ cells mL⁻¹, but had little stimulatory effect on P. donghaiense at a density of 1.9 × 10⁵ cells mL⁻¹compared to the control (P > 0.05). It is likely that these two species of microalgae interact with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. We then quantify their interactions in the bi-algal culture by using a mathematical model. The estimated parameters from the model showed that the inhibition exerted by S. trochoidea on P. donghaiense was about 43 and 24 times stronger than the inhibitory effect that P. donghaiense exerted on S. trochoidea when the initial size/density were 1:1 and 10:1, respectively. S. trochoidea seemed to have a survival strategy that was superior to P. donghaiense in the bi-algal culture under controlled laboratory conditions. We also observed a closely positive relationship between the initial cell density and its effect on the co-cultured microalga by measuring the fluorenscence: filtrate prepared from higher initial cell density had stronger interference on the co-cultured microalga. Moreover, pre-treated under different temperature conditions (30 °C, 60 °C and 100 °C) would significantly changed the effect of culture filtrate on the co-cultured microalga. Result inferred that P. donghaiense or S. trochoidea would release allelochemicals into the bi-algal culture medium and the allelochemicals might be a mixture with temperature-sensitive components in it.