Drag-reducing polysaccharides from marine microalgae: species productivity and drag reduction effectiveness

We have conducted a study of the potential use of drag-reducing biopolymers produced by marine microalgae for engineering applications. Several marine microalgae species were tested for their production of drag-reducing polysaccharides in large custom-designed plate bioreactors. Promising species (such as Porphyridium cruentum, Rhodella maculata, Schizochlamydella capsulata and Chlorella stigmatophora) were cultured for periods of time ranging from a few weeks to over 6 months. The basic drag-reducing ability of the polysaccharides was established by comparing their drag reduction effectiveness at various concentrations in water. The algal polysaccharide mass productivity was also measured per unit area of bioreactor’s illuminated surface. Finally, an all-inclusive criterion, the volumetric production of drag-reducing water giving a set level of drag reduction was quantified, and led us to a ranking of the tested species in order of productivity relevant to implementation. Some aspects of polysaccharide production by aged cultures were investigated as well. We also quantified the drag-reducing effectiveness of intracellular polysaccharides, and visualized the presence of exopolymer particles in the medium.     

Purification and Chemical Properties of Acinetobacter calcoaceticus A2 Biodispersan

The extracellular dispersant of Acinetobacter calcoaceticus A2, referred to as biodispersan, was concentrated by ammonium sulfate precipitation and deproteinized by hot phenol treatment. The active component was an anionic polysaccharide (PS-A2). The specific activity of PS-A2 was approximately three times greater than that of crude biodispersan. PS-A2 had a sedimentation constant of 1.39 S, a diffusion coefficient of 18.8 × 10⁻⁸ cm² s⁻¹, and a partial molar volume of 0.65 cm³ g⁻¹, yielding an average molecular weight of 51,400. Titration of the polymer gave two inflection points: pK₁ = 3.1 (1.15 μEq/mg) and pK₂ = 8.0 (0.4 μEq/mg). PS-A2 slowly consumed 1.10 μmol of periodate per mg. The ¹³C nuclear magnetic resonance spectrum of PS-A2 indicated four methyl groups, four carbonyl C atoms, and four signals in the anomeric region (95 to 110 ppm), indicative of the presence of four different monosaccharides. Strong acid hydrolysis of PS-A2 yielded four reducing sugars: glucosamine, a 6-methyl aminohexose, galactosamine uronic acid, and an unidentified amino sugar. Ruthenium red binding to PS-A2 was stoichiometric: 1 molecule of dye bound per 2.0 carboxyl groups.     

Induction of Heat Stress Tolerance in Barley Seedlings by Pre-Sowing Seed Treatment with Glycinebetaine

Heat stress adversely affects plant growth and development, while glycinebetaine (GB) plays a protective role under stressful conditions. The objective of this study was to assess the optimum level of GB for use as a presowing seed treatment and the subsequent effect on the heat tolerance of barley (Hordeum vulgare L. cv. Haider-93) seedlings. Among a range of GB levels, the 20 mM concentration emerged as the most effective in enhancing seed germination, shoot fresh and dry weight and shoot water content under heat stress, and this level was selected for further studies. Time course changes revealed that the seedlings developing from 20 mM GB treated seeds had greater shoot dry weight, net photosynthetic rate (P_N), leaf water potential (ψ_w) and reduced relative membrane permeability (RMP), compared to no-GB treated plants under heat stress. Correlations between dry weight and high P_N (r = 0.881), low ψ_w (r = −0.938) and RMP (r = −0.860) of shoots suggested the involvement of GB in heat stress tolerance. Leakage of Ca²⁺ and NO₃⁻ was the greatest followed by K⁺ and PO₄³⁻ under no-GB seed treatment, and GB application under heat stress appreciably reduced the leakage of all these ions, particularly Ca²⁺, K⁺ and NO₃⁻. In conclusion GB absorbed by seeds, after translocation to the seedlings, enhanced their capacity to maintain greater water content, and higher seedling vigor by virtue of increased P_N, reduced RMP and leakage of important ions under heat stress. These results have implications for final field stand under the conditions where the ambient temperature is supra-optimal for barley growth.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

Overproduction of an extracellular polysaccharide possessing high lipid emulsion stabilizing effects by Bacillus sp.

Bacillus sp. CP912, producing an extracellular biopolymer, was isolated from the soil. Maximum accumulation of the biopolymer was 10 g l^–1 culture broth with a yield of 88% from glucose consumed. The biopolymer was purified with several precipitation steps using ethanol and cetyl-trimethyl-ammonium bromide. Carbohydrate analyses using various color reactions, infrared spectroscopy, and high performance liquid chromatography revealed that the biopolymer is a homopolysaccharide. The lipid emulsifying capacity of the polysaccharide was 100%, while that of xanthan gum was 94%.     

Polysaccharide synthesis by Phanerochaete chrysosporium during degradation of kraft lignin

Summary Phanerochaete chrysosporium (Sporotrichum pulverulentum) produced an extracellular glucan type polysaccharide when grown in a chemostat under nitrogen limitation. When cells were transferred to a standing mode of cultivation in the presence of excess glucose (6 gl^–1), the amount of non-glucose total carbohydrates in the culture increased from 0.58 gl^–1 to 1.76 gl^–1 during 15 day experiments. The change in total carbohydrates was due to an increase in extracellular and cell-bound glucan type polysaccharide. This increase occured simultaneously with formation of mycelial mats and appearance of ligninolytic activity. When the cultures were agitated under atmospheric oxygen rather than 100% O₂, their non-glucose total carbohydrate content increased to 2.15 gl^–1 in 4 days. The excess polysaccharide formation had an inhibitory effect on lignin degradation as more lignin was degraded by cells with lower polysaccharide content. The lignin that was associated with cells after the degradation had stopped could be further degraded by new active cells.     

The endogenous metabolism ofFibrobacter succinogenes and its relationship to cellobiose transport,viability and cellulose digestion

Fibrobacter succinogenes S85 digested ballmilled cellulose at a rapid rate (0.10 h^–1), but there was a long lag time if the culture was not transferred daily. WhenF. succinogenes was starved for 100h, a large fraction of the cells (>30%) still bound to cellulose, but the lag time was 150h. The lag time was similar for either cellulose- or cellobiose-grown inocula, and lag times were highly correlated (r ² = 0.91) with a decrease in viable cell number. The number of viable cells declined from 10⁸ to 10⁶ in the first 30h of starvation, and by 72h the viable cell number was less than 10³/ml. Cells growing exponentially on cellobiose had a large pool of polysaccharide, and continuous culture experiments indicated that polysaccharide accumulation was not significantly influenced by the growth rate of the culture (approximately 0.7 mg polysaccharide mg^–1 protein). When the cellobiose was depleted, cellular polysaccharide decreased at first order rate of 0.09 h^–1. The rate of endogenous metabolism was initially 0.08mg polysaccharide mg^–1 protein h^–1, and there was little decline in viability until the rate of endogenous metabolism was less than 0.01 mg polysaccharide mg^–1 protein h^–1. When the rate was less than 0.01 mg polysaccharide mg^–1 protein h^–1, the cells could not maintain a sodium gradient, transport cellobiose or grow. The endogenous metabolic rate needed for cell survival was 20 fold less than the maintenance energy of cells growing in continuous culture (0.01 versus 0.232mg carbohydrate mg^–1 protein h^–1).     

Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions

The concentration (c) and shear rate (γ) dependence of viscosity (η) has been studied for a wide range of random coil polysaccharide solutions, and the following striking generalities are observed:

1. 1. The transition from dilute to concentrated solution behaviour occurs at a critical concentration , when ‘zero shear’ specific viscosity (η_sp) ≈ 10. η_sp varies as c^1.4 for dilute solutions, and as c^3.3 for concentrated solutions.

2. 2. The shear rate dependence of viscosity, and frequency dependence of dynamic (oscillatory) viscosity are closely superimposable.

3. 3. Double logarithmic plots of against (where η₀ is ‘zero shear’ viscosity, and is the shear rate at which ) are essentially identical for all concentrated solutions studied, and thus the two parameters η₀ and completely define the viscosity at all shear rates of practical importance.

Departures from points 1 and 2, but not 3, are observed for concentrated solutions of locust bean gum, guar gum, and hyaluronate at low pH and high ionic strength and are attributed to specific intermolecular associations (‘hyperentanglements’) of longer timescale than non-specific physical entanglements.     

Bacteria Associated with Mucus and Tissues of the Coral Oculina patagonica in Summer and Winter

The relative abundance of bacteria in the mucus and crushed tissue of the Mediterranean coral Oculina patagonica was determined by analyses of the 16S rRNA genes of isolated colonies and from a 16S rRNA clone library of extracted DNA. By SYBR gold staining, the numbers of bacteria in mucus and tissue samples were 6.2 × 10⁷ and 8.3 × 10⁸/cm² of coral surface, respectively, 99.8% of which failed to produce colonies on Marine Agar. From analysis of mucus DNA, the most-abundant bacterium was Vibrio splendidus, representing 68% and 50% of the clones from the winter and summer, respectively. After removal of mucus from coral by centrifugation, analyses of DNA from the crushed tissue revealed a large diversity of bacteria, with Vibrio species representing less than 5% of the clones. The most-abundant culturable bacteria were a Pseudomonas sp. (8 to 14%) and two different α-proteobacteria (6 to 18%). Out of a total 1,088 16S rRNA genes sequenced, 400 different operational taxonomic units were identified (>99.5% identity). Of these, 295 were novel (<99% identical to any sequences in the GenBank database). This study provides a comprehensive database for future examinations of changes in the bacterial community during bleaching events.     

Biosurfactant from <Emphasis Type="Italic">Lactococcus lactis</Emphasis> 53 inhibits microbial adhesion on silicone rubber

The ability of biosurfactant obtained from the probiotic bacterium Lactococcus lactis 53 to inhibit adhesion of four bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber with and without an adsorbed biosurfactant layer was investigated in a parallel-plate flow chamber. The microbial cell surfaces and the silicone rubber with and without an adsorbed biosurfactant layer were characterized using contact-angle measurements. Water contact angles indicated that the silicone-rubber surface with adsorbed biosurfactant was more hydrophilic (48°) than bare silicone rubber (109°). The results showed that the biosurfactant was effective in decreasing the initial deposition rates of Staphylococcus epidermidis GB 9/6 from 2,100 to 220 microorganisms cm^–2 s^–1, Streptococcus salivarius GB 24/9 from 1,560 to 137 microorganisms cm^–2 s^–1, and Staphylococcus aureus GB 2/1 from 1,255 to 135 microorganisms cm^–2 s^–1, allowing for a 90% reduction of the deposition rates. The deposition rates of Rothia dentocariosa GBJ 52/2B, Candida albicans GBJ 13/4A, and Candida tropicalis GB 9/9 were far less reduced in the presence of the biosurfactant as compared with the other strains. This study constitutes a step ahead in developing strategies to prevent microbial colonization of silicone-rubber voice prostheses.     

Small-scale turbulence affects the division rate and morphology of two red-tide dinoflagellates

The effects of small-scale turbulence on two species of dinoflagellates were examined in cultures where the turbulent forces came randomly from all directions and were intermittent both spatially and temporally; much like small-scale turbulence in the ocean. With Lingulodinium polyedrum (Stein) Dodge (syn. Gonyaulax polyedra), division rate increased linearly (from 0.35 to 0.5 per day) and the mean cross-sectional area (CSA) decreased linearly (from 1100 to 750 μm²) as a function of the logarithmic increase in turbulence energy dissipation rate (). These effects were noted when values increased between 10⁻⁸ and 10⁻⁴ m² s⁻³. However, when increased to 10⁻³ m² s⁻³, division rate sharply decreased and mean CSA increased. Over the same range of , Alexandrium catenella (Wheedon and Kofoid) Balech had its division rate decrease linearly (from 0.6 to 0.45 per day) and its CSA increase linearly (from 560 to 650 μm²) as a function of the logarithmic increase in . Even at the highest examined (10⁻³ m² s⁻³), which may be unrealistically high for their ambits, both L. polyedra and A. catenella still had fairly high division rates, 0.2 and 0.45 per day, respectively. Turbulence strongly affected chain formation in A. catenella. In non-turbulent cultures, the mode was single cells (80–90% of the population), but at of 10⁻⁵ to 10⁻⁴ m² s⁻³, the mode was 8 cells per chain. At the highest (10⁻³ m² s⁻³), the mode decreased to 4 cells per chain. The vertical distributions of A. catenella populations in relation to hydrographic flow fields were studied in the summers of 1997 and 1998 in East Sound, Washington, USA (latitude 48°39′N, 122°53′W). In both summers, high concentrations of A. catenella were found as a subsurface bloom in a narrow depth interval (2 m), where both current shear and turbulence intensity were at a minimum. Other researchers have shown that A. catenella orients its swimming in shear flows, and that swimming speed increases with chain length. These responses, when combined with our observations, support a hypothesis that A. catenella actively concentrates at depths with low turbulence and shear.     

Production of a hypoglycemic, extracellular polysaccharide from the submerged culture of the mushroom, Phellinus linteus

Mycelial growth and extracellular polysaccharide production of Phellinus linteus were optimal at pH 5 and 25 °C. Maximum biomass production (14.2 g l^–1) was after 15 d of cultivation, whereas, extracellular polysaccharide was maximal (3.5 g l^–1) after 21 d. The hypoglycemic effect of the polysaccharide, investigated in streptozotocin-induced diabetic rats, decreased plasma glucose, total cholesterol and triacylglycerol concentrations by 49%, 32%, and 28%, respectively, and aspartate aminotransferase activity by 20%. The results indicate the potential of this polysaccharide to prevent hyperglycemia in diabetic patients.     

Coral-crab association: a compact domain of a multilevel trophic system

Colonies of the Red Sea branching coral Stylophora pistillata were incubated in situ with radioactive carbon and Trapezia cymodoce crabs were introduced to the colonies, for one month each, up to 7 months after coral labelling. Zooxanthellar photosynthetic products were translocated to the crabs via host coral tissue. Based upon crab/coral tissue conversion from 53 crab samples, crabs possessed radioactive material equal to that recorded in 320–770 mm² of coral tissue (up to 2257 mm²). This material was translocated mainly by direct grazing on coral tissue rather than mucus collection. Ovigerous female crabs (39 specimens) accumulated significantly more labelled carbon than males, and up to 53 % of their radioactivity was concentrated in their reproductive organs. A pair of crabs dwelling in a coral colony consumed ca 130 cm² of host tissue per month (40–45 cm length of coral branches). This system represents a compact, obligatory multilevel trophic domain which also radiates horizontally by allocation of energy derived from algal photosynthesis into planula and zooea larva production, permitting the development of long trophic chains and a complex food web.     

Enhanced shear protection and increased production of an anti-tumor polysaccharide by Agaricus blazei in xanthan-supplemented cultures

Xanthan supplementation provided shear protection and stimulated polysaccharide production by Agaricus blazei. In xanthan-free cultures, the optimal cell yield, 0.63 g biomass g^–1 glucose, and product yield, 0.19 g polysaccharide g^–1 glucose, were, respectively, when the critical impeller tip speed was 50.3 cm s^–1 and 100.5 cm s^–1. Furthermore, the critical impeller tip speed of cell yield shifted from 50.3 cm s^–1 to 100.5 cm s^–1 with the supplementation of 1 g xanthan l^–1. Maximum specific product yield, namely 0.74 g polysaccharide g^–1 biomass, was achieved with inlet air supply of 3% O₂ and impeller tip speed of 100.5 cm s^–1.     

Localization of Acetyl Groups in the Macromolecule of Glucomannan Obtained from Roots of Eremurus zangezuricus

Water-soluble glucomannan from roots of Eremurus zangezuricus Mikheev was studied. The polysaccharide contains D-glucose, D-mannose, and acetyl groups in the molecular ratio of 1 : 2.8 : 0.38. ¹³C NMR studies showed that the polysaccharide under study is a linear, partly acetylated 1,4--D-glucomannan. The acetyl groups are attached to the C2 and C3 of mannopyranose units. The polymer contains, on average, one acetyl group per seven mannose units. The glucomannan of E. zangezuricus has the following parameters: []_D = –37.5°, [] = 3.73 dl/g, and Mw = 151 kDa.     

Fish skin bacteria: Production of friction-reducing polymers

The supernatant fluids of cultures of four bacterial strains isolated from the skin of barracuda contained extracellular polymer concentrations of 0.2–0.5 mg/ml and reduced factional drag by 2.5–22% in a turbulent flow rheometer. The production and properties of one of the drag-reducing polymers, referred to as PS-6, were studied further. Polymer PS-6 was produced by strain NS-31 in minimal salts medium supplemented with ethanol or glucose. The polymer began to accumulate in the culture medium during exponential phase and continued to be produced during stationary phase. It reduced drag by 55% at a concentration of 0.75 mg/ ml. Acid-base titration of the deproteinized polymer PS-6A gave two inflection points: pK₁=3.26 (2.4 eq/mg) and pK₂=9.66 (0.8 eq/mg).¹³C-NMR spectroscopy of PS-6A resolved 25 peaks, including three methyl groups, three carbonyl groups, and four signals in the anomeric region (99–103 ppm), indicating the presence of four different monosaccharides. Strong acid hydrolysis of PS-6A yielded an amino acid, pyruvate, and four reducing sugars: a hexosamine, a uronic acid, and two hexoses which migrated on TLC similarly to glucose and galactose. The possible role of bacteria in production of drag-reducing mucus is discussed.     

An extracellular sulfated fucose-rich polysaccharide produced by a tropical strain of Cryptomonas obovata (Cryptophyceae)

A tropical strain of Cryptomonas obovata Skuja, isolated from a shallow oxbow lake,releaseda sulfated fucose-rich polysaccharide. The polysaccharide is composed mainly offucose (42%), N-acetyl-galactosamine (26%) and rhamnose (15%), with smallquantities of glucuronic acid, mannose, galactose, xylose and glucose. Sulfateaccounted for 1.7% total polysaccharide. Quantitative release was studied withcells exposed to optimal culture conditions contrasted with high irradiance andnitrate depletion. This latter set of conditions could simulate stresssituations usually found in the place from which this strain was isolated. Themonosaccharide composition of the polysaccharide was evaluated using PAD-HPLCand gas chromatography. The two irradiances tested (165 molm^–2 s^–1 and 2000 molm^–2 s^–1) had no significant effect onamounts of polysaccharide released by the cells. Differences were observed whenthe nitrate availability was varied. In the nitrate-depleted situation,extracellular polysaccharide production was 2.5 times higher than replete cellsafter 6 h at 165 mol m^–2s^–1, and 2.25 times higher at 2000 molm^–2 s^–1.     

Cultivation ofMonostroma nitidum (Chlorophyta) in a river estuary,southern Japan

The green seaweedMonostroma nitidum Wittrock is cultivated in brackish waters of southern Japan and an ecological survey of its cultivation was carried out in the estuary of R. Shimanto over three years. Artificial seed culture began by collecting many gametes in April. The zygotes adhered to a plastic settlement board (20–30 cm long and 10 cm wide). The cultivated zygotes in the indoor tank increased gradually in size from 8 to 40m till early September. Maturation of the zygote was promoted by providing dark conditions for 2–3 weeks. The production of a concentrated zoospore solution was achieved by adding freshwater 2–3°C above that of the zygote culture tank and a photon flux density of 100mol m^–2 s^–1).The culture nets were set our horizontally at a level exposed for 4 h and were harvested 3–4 times till March. The total production was approximately 6–10 kg dry weight per net during culture periods. The total production of nets harvested 3–4 times per year was greater than that of nets harvested only once.     

Effects of photon flux density,CO2, aeration rate,and inoculum density on growth and extracellular polysaccharide production byPorphyridium cruentum

Growth and extracellular polysaccharide production byPorphyridium cruentum were measured as a function of several culture parameters. Photon flux density of 75 μmol m⁻² s⁻¹ and CO₂ concentration of 2.5% were found to be optimum for both growth and extracellular polysaccharide production. Interactive studies on these two parameters further confirmed that at these levels of photon flux density and CO₂, when applied together, both growth (5.9·10⁷ cells per mL) and extracellular polysaccharide production (1.9 g/L) were at the maximum. Maximum growth and extracellular polysaccharide production were observed at inoculum density of 10⁶ cells per mL and aeration rate of 500 mL air per min per liter.     

Brachionus calyciflorus Pallas as agent for the removal of E. coli in sewage ponds

Brachionus calyciflorus (Pallas) is a common brachionid in sewage oxidation ponds. The uptake and assimilation of E. coli was optimal at concentrations of 2.7–6.9 × 10⁸ cells ml^–1 while assimilation coefficient per body weight of B. calyciflorus was found to be 10% · Ind.^–1 d^–1. More than two eggs per individual were produced during 24 hours when brachionids were fed with a mixutre of E. coli (10⁹ cells · ml^–1) and Chlorella spp. (10⁶ cells · ml^–1). The nutritional value of the mixture of E. coli and Chlorella spp. was found to be higher than that of bacteria alone.