Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater

Aims: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. Methods and Results: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38°C for growth and inulinase production, and 35°C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l^?1, and the highest ethanol yield of 0·467, or 91·5% of the theoretical value of 0·511, was achieved for the slurry with a solid concentration of 200 g l^?1. Conclusions: Halophilic Jerusalem artichoke can be used for fuel ethanol production. Significance and Impact of the Study: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.     

Effects of deep seawater medium on growth and amino acid profile of a sterile Ulva pertusa Kjellman (Ulvaceae, Chlorophyta)

The potential use of deep seawater (DSW) for the cultivation of macroalgae was evaluated by analysis of the growth rate of thalli (by surface area), photosynthetic pigment content, and total free amino acid content of sterile Ulva pertusa cultured in the control medium (one-fifth strength Provasoli’s enriched seawater (PES) medium), surface seawater (SSW) medium and also seawater medium collected from 400 m depth (400-DSW) and 1,200 m depth (1,200-DSW). The growth rate of the sterile U. pertusa grown in 400-DSW and 1,200-DSW was similar to that of thalli cultured in the one-fifth strength PES medium that had previously been shown to be the optimum concentration for the cultivation of Ulva. Photosynthetic pigment content in the control medium, 400-DSW- and 1,200-DSW-cultured thalli was 1.7–2.0 times higher than that of the SSW-cultured thalli. The total free amino acids content of the control medium and SSW-cultivated thalli was 264?±?64 and 120?±?39 mg per 100 g dry weight, whereas the 400-DSW- and 1,200-DSW-cultivated thalli had significantly more amino acids (342?±?69–327?±?64 mg per 100 g dry weight). The results of this study indicate that DSW has a high potential for cultivation of edible macroalgae without any supply of extra nutrient until it grows to harvest size.     

Activation of carbonic anhydrase in branchial cavity tissues of lobsters (Homarus gammarus) by dilute seawater exposure

Activities of carbonic anhydrase (CA) and its distribution in the branchial cavity tissues were studied in European lobsters (Homarus gammarus) from ambient seawater (SW; salinity=38 ppt, 1126 mosmol/l) and acclimated to dilute seawater (DSW; salinity=20 ppt, 548 mosmol/l). Acetazolamide inhibited dose dependently the activity of CA in homogenates of epipodites, where the inhibition constant (IC₅₀=0.12 μM) did not differ significantly from that of membrane vesicles and cytosolic fraction. In DSW-acclimated lobsters, almost 70% of total CA in tissues of the branchial cavity was found in epipodites (E) and the rest was equally distributed between branchiostegites (B) and gills (G). Upon acclimation to dilute seawater, CA activity in membrane fractions of E and B was increased 6-fold and in homogenates, respectively 5- and 13-fold compared to SW-acclimated lobsters. Exposure to DSW enhanced cytosolic CA in E (8-fold) and B (7-fold) over SW-acclimated animals. Slight activation of CA in homogenates and in partially purified membranes of G was not confirmed as a statistically significant difference between SW and DSW groups. In DSW, cytosol specific activity of CA was increased compare to the SW cytosol. These results indicate the importance of E and B in CA induction when lobsters are acclimated to DSW. In subcellular fractions from DSW-acclimated lobsters, the main proportion of 75.8% (E), 61.0% (B) of total CA activity in each of these tissues remained in cytosol portion. Partially purified membranes contained 6.8% (E) and 16.2% (B) and the remainder of 15% (E) and 27% (B) was found in mitochondrial and nuclear fractions. In gills, 49.2% and 9.0 % of total gill CA activity was found respectively in cytosol and partially purified vesicles and the rest in mitochondrial and nuclear fractions.     

Changes in Na+/K+-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation

We have assessed the activity of Na⁺/K⁺-ATPase, cAMP, free fatty acids (FFA) and metallothionein (MT) in the posterior gills of the brackish water shore crab Carcinus aestuarii during acclimation to 10 ppt dilute seawater (DSW). Following 3–18 days acclimation in DSW specific activity of Na⁺/K⁺-ATPase in native gill homogenates and partially purified membrane vesicles was progressively increased, from 1.7- to 3.9-fold. After short-term acclimation of crabs in DSW with added sucrose to make media isosmotic with the haemolymph the specific Na⁺/K⁺-ATPase activity in homogenates was not increased, relative to SW enzyme activity. Moreover, hyposmotic conditions led to depletion of cAMP in gills.In partially purified membrane vesicles isolated from posterior gills, fatty acids with compositions 16:0, 18:0, 18:1, 20:4 and 20:5 dominated in both SW- and DSW-acclimated Carcinus. During a year in which the metabolic activity of crabs was increased, the arachidonic/linoleic acids ratio (ARA/LA) for DSW-acclimated crabs was markedly increased relative to that in SW. Increased Na⁺ + K⁺-ATPase activity under hyposmotic stress may be modulated at least partially by the changed proportion of fatty acids in the purified membranes of posterior gills. Long-term acclimation of shore crabs to DSW resulted in a 2.6-fold increase in cytosolic metallothionein (MT) content in posterior gills over those in SW crabs. Assuming an antioxidant role of MT associated with intracellular zinc partitioning, the observed MT induction in posterior gills may be considered an adaptive response of C. aestuarii to hyposmotic stress.     

Tank cultivation of <Emphasis Type="Italic">Ulva prolifera</Emphasis> in deep seawater using a new “germling cluster” method

A new “germling cluster” method is proposed for tank cultivation of seaweed in a free-floating form. This method was applied to the tank cultivation of Ulva prolifera using deep seawater (DSW) pumped up from over 300 m depth off the cape of Muroto in southwest Japan. Numerous zoids of U. prolifera were induced by cutting thalli into 1–2 mm long pieces. Three days after fragment production, the zoids were released. The zoid suspension was concentrated to a density of more than 10⁴ zoids per mL medium, and placed in a Petri dish for culture. The dense, germinating zoids began to adhere to each other and form aggregations. The germling aggregations were then removed from the bottom of the dish and torn into a large number of small “germling clusters” using an electric mixer. Each cluster contained 10–100 germlings. Once the germling clusters had attained more than 5 mm diameter in culture, they were transplanted as free-floating forms to a 500 L outdoor tank with continuous aeration, to which DSW was supplied at an exchange rate of 3 volumes per day. As a result, the average daily growth rate (DGR) in the tank throughout the year was 37%, though the DGR fluctuated with seasonal temperature changes.     

Changes in the composition of coelomic fluid in the Atlantic stingray,Hypanus sabina,upon dilution of the environmental salinity: osmoregulatory and pH implications

Ichthyological Research - Euryhaline stingrays, Hypanus sabina, adapted to full-strength seawater (FSW) were transferred to diluted (50%) seawater (DSW). Osmolytes in plasma (PL), coelomic fluid...     

Stimulative and inhibitory effects of bacteria on the growth of microalgae

Several examples of stimulative and inhibitoryeffects of bacteria on microalgal growth areintroduced, and the importance of bacteria in algalmass culture is investigated. Diatoms are often usedas live food for planktonic larvae of sea urchin andbivalves. Monodispersed Chaetoceros ceratosporum hasbeen cultivated by using clean, high nutrient content,deep seawater (DSW). However, the growth rate and cellyield of diatoms fluctuated, to relatively largeextent, with the season that DSW was collected. Whensome bacterial strains isolated from DSW were added tothe culture, diatom growth was often stimulated and arelatively constant cell yield was obtained. Anotherdiatom species, C. gracilis, was also stimulated byadding some bacterial strains to cultures. Thepositive effect of bacteria on diatoms was observednot only for planktonic species, but also on attachedspecies. A benthic diatom, Nitzschia sp., wasstimulated by a bacterial film of Alcaligenes on thesurface of the substratum. On the other hand, a strainof Flavobacterium sp. isolated from natural seawaterduring the decline period of an algal bloom had a strongalgicidal effect on the red tide plankton,Gymnodinium mikimotoi. Recent reports demonstratethat many bacterial strains have significantalgicidal effects on many species of red tideplankton. These results indicate that bacterialeffects should be taken into account to obtain stablemass culture of food microalgae.     

Intake of dissolved organic matter from deep seawater inhibits atherosclerosis progression

Dissolved organic matter (DOM) in seawater can be defined as the fraction of organic matter that passes through a filter of sub micron pore size. In this study, we have examined the effect of DOM of deep seawater (DSW) from Pacific Ocean on platelet aggregation and atherosclerosis progression. DSW was passed through a series of filters and then through an Octadecyl C18 filter; the retained substance in ethanol was designated as C18 extractable DOM (C18-DOM). Our studies showed that C18-DOM treatment inhibited platelet aggregation, P-selectin expression and activity of COX-1 significantly. C18-DOM increased the expression of anti-atherogenic molecule namely heme oxygenase-1 in endothelial cells and all these data showed that C18-DOM is exhibiting aspirin-like effects. Moreover our in vivo studies showed that C18-DOM feeding slowed remarkably the progression of atherosclerosis. Our study demonstrated a novel biological effect of oceanic DOM, which has several important implications, including a possible therapeutic strategy for atherosclerosis.     

Continuous culture with deep seawater of a benthic food diatom Nitzschia sp.

A continuous culture system for a benthic food diatom Nitzschia sp. wasestablished by using properties of high nutrient and clean of deep seawater(DSW). DSW collected from 320 m depth in Muroto City, Japan, was introducedinto a glass-pipe bioreactor (14 cm length, 3 cm diam.) containing glassbeads of 0.5 cm diam. as substrata for the alga, and it was incubated at18°C · 80Em^–2sec^–1 · L:D=14:10. The chlorophyll a yield of benthicdiatoms in a reactor as a unit of surface area of the substratum was only0.001–0.003 g cm^–2 when the flow rate of DSW was 0(batch culture conditions). However, when DSW was supplied continuously to areactor, the yield increased to 1.4 g-chl.a cm^–2 alongwith the increase in flow rate of DSW. Moreover, amounts of chl.a washed outof the system were negligible, 0.0014 to 0.0045%, even though theflow rate of DSW was as much as 25 times h^–1, suggesting thatsloughing of benthic diatoms from the substratum was minimized. Although theyield of diatoms fluctuated significantly at the time that the DSW wascollected, the variation could be minimized by increasing the flow rate ofDSW. These results indicate that the continuous culturing system with DSWsupports the stable and effective mass culture of benthic food diatom.     

Deep-Sea Water Containing Selenium Provides Intestinal Protection against Duodenal Ulcers through the Upregulation of Bcl-2 and Thioredoxin Reductase 1

Deep-sea water (DSW), which is rich in micronutrients and minerals and with antioxidant and anti-inflammatory qualities, may be developed as marine drugs to provide intestinal protection against duodenal ulcers. We determined several characteristics in the modified DSW. We explored duodenal pressure, oxygenation, microvascular blood flow, and changes in pH and oxidative redox potential (ORP) values within the stomach and duodenum in response to tap water (TW, hardness: 2.48 ppm), DSW600 (hardness: 600 ppm), and DSW1200 (hardness: 1200 ppm) in Wistar rats and analyzed oxidative stress and apoptosis gene expressions by cDNA and RNA microarrays in the duodenal epithelium. We compared the effects of drinking DSW, MgCl₂, and selenium water on duodenal ulcers using pathologic scoring, immunohistochemical analysis, and Western blotting. Our results showed DSW has a higher pH value, lower ORP value, higher scavenging H₂O₂ and HOCl activity, higher Mg²⁺ concentrations, and micronutrients selenium compared with TW samples. Water infusion significantly increased intestinal pressure, O₂ levels, and microvascular blood flow in DSW and TW groups. Microarray showed DSW600, DSW1200, selenium water upregulated antioxidant and anti-apoptotic genes and downregulated pro-apoptotic gene expression compared with the TW group. Drinking DSW600, DSW1200, and selenium water but not Mg²⁺ water significantly enhanced Bcl-2 and thioredoxin reductase 1 expression. Bax/Bcl-2/caspase 3/poly-(ADP-ribose)-polymerase signaling was activated during the pathogenesis of duodenal ulceration. DSW drinking reduced ulcer area as well as apoptotic signaling in acetic acid-induced duodenal ulcers. DSW, which contains selenium, provides intestinal protection against duodenal ulcers through the upregulation of Bcl-2 and thioredoxin reductase 1.     

Anti-obesity and Antidiabetic Effects of Deep Sea Water on ob/ob Mice

Recently, deep sea water (DSW) has started to receive much attention for therapeutic intervention in some lifestyle diseases. In this study, the anti-obesity and antidiabetic effects of DSW in ob/ob mice were investigated. The animals were randomly divided into two groups with six animals: control group received tap water; the experimental group was treated with DSW of hardness 1000 for 84 days. The body weight gain after 84 days in DSW-fed group was decreased by 7% compared to the control group. The plasma glucose levels in the DSW-fed mice were substantially reduced by 35.4%, as compared to control mice. The results of oral glucose tolerance test revealed that DSW-fed groups significantly increased the glucose disposal after 84 days. DSW increased plasma protein levels of adiponectin and decreased plasma protein levels of resistin, RBP4, and fatty acid binding protein. Moreover, GLUT4 and AMP-activated protein kinase levels in skeletal muscle tissue were increased while peroxisome proliferator-activated receptor γ and adiponectin were decreased in adipose tissue of DSW-fed mice. These results suggest that the antidiabetic and anti-obesity activities of DSW were mediated by modulating the expression of diabetes- and obesity-specific molecules. Taken together, these results provide a possibility that continuous intake of DSW can ameliorate obesity and diabetes.     

Effect of parental mate choice and semi‐natural early rearing environment on the growth performance and seawater tolerance of Chinook salmon Oncorhynchus tshawytscha

To assess whether parental mate choice and early rearing in a semi‐natural spawning channel may benefit the culture of Chinook salmon Oncorhynchus tshawytscha, 90 day growth trials were conducted using hatchery O. tshawytscha (hatchery), mate choice O. tshawytscha (i.e. the offspring of parents allowed to choose their own mate) that spent 6 months in a spawning channel prior to hatchery rearing (channel) and mate choice O. tshawytscha transferred to the hatchery as fertilized eggs (transfer). During the growth trials, all O. tshawytscha stocks were reared separately or in either mixed channel and hatchery or transfer and hatchery groups for comparison of performance to traditional practices. After 60 days in fresh water, all O. tshawytscha were transferred to seawater for an additional 30 days. Reared separately, all stocks grew c. 4·5 fold over 90 days but specific growth rate (G) and food conversion efficiency were higher in fresh water than after seawater transfer on day 60. In contrast, hatchery O. tshawytscha from mixed hatchery and channel and hatchery and transfer growth trials had a larger mass and length gain than their counterparts on day 60, but reduced G in seawater. In general, plasma levels of growth hormone, insulin‐like growth factor I and cortisol did not differ among any O. tshawytscha groups in either the separate or mixed growth trials. Despite some differences in gill Na⁺,K⁺‐ATPase activity, all O. tshawytscha had a high degree of seawater tolerance and experienced virtually no perturbation in plasma chloride following seawater transfer. Overall, all O. tshawytscha exhibited similar growth and seawater performance under traditional hatchery conditions and any benefit derived from either parental mate choice or semi‐natural early rearing environment was only observed in the presence of mutual competition with hatchery O. tshawytscha.     

不同食物及食物密度对盐蚕豆虫 种群动力学特征的影响

为探究不同食物及食物密度对盐蚕豆虫（Fabrea salina）种群动力学的影响。本文采用商业酵母和杜氏藻（Dunaliella salina）两种不同类型的饲料喂食盐蚕豆虫,探究其种群动力学特征。结果表明,杜氏藻组的盐蚕豆虫具有较高的生长率[（0.78 ± 0.019）/d]、较低的世代时间[（0.89 ± 0.021）d],内禀生长率为1.49/d,米氏常数值为1 121.32;商业酵母组生长率较低[（0.36 ± 0.001）/d],世代时间较长[（1.93 ± 0.007）d],内禀生长率为0.51/d,米氏常数值为2.68。One-way ANOVA检验结果表明,食物及食物密度对盐蚕豆虫的种群生长均有极显著影响（P < 0.01）。以密度为10 × 109 cells/L的杜氏藻投喂,可在短时间内实现盐蚕豆虫的高密度培养和利用,而酵母适用于实验室中盐蚕豆虫的保种工作。     

Seawater Stress Differentially Affects Germination, Growth, Photosynthesis, and Ion Concentration in Genotypes of Jerusalem Artichoke (Helianthus tuberosus L.)

Two Jerusalem artichoke (Helianthus tuberosus L.) genotypes, NY-1 and NY-7, were subjected to different seawater concentrations (0, 10, 20, 30, 40, and 50%) for various periods of time to determine the effects on seedling growth, ion content, and photosynthetic productivity in a greenhouse. Under different seawater concentrations, sprouting rates varied greatly among the genotypes. The differences in relative growth rate (RGR), leaf chlorophyll content, total leaf area (TLA), plant dry weight (PDW), photosynthetic rate (A), stomatal conductance (g _s), and efficiency of the light harvesting of photosystem II (F _v^′/F _m^′) were significant between NY-1 and NY-7 after 12 days of stress at 40 and 50% seawater. Seawater treatments resulted in the reduction of almost all the growth parameters and coincident increases of Na⁺ and Ca²⁺ concentrations in plant tissues. Our results indicate that there is great variability for seawater tolerance among H. tuberosus varieties, and that greater photosynthesis capacity, higher RGR, and relatively higher tissue Na⁺ accumulation at high seawater concentrations appears to be associated with seawater tolerance in H. tuberosus varieties.     

Gill Na,K-ATPase in the spiny lobster Palinurus elephas and other marine osmoconformers. Adaptiveness of enzymes from osmoconformity to hyperregulation

Haemolymph inorganic osmolyte changes and Na,K-ATPase activities in trichobranchiate and epipodite tissues were examined in the spiny lobster Palinurus elephas gradually acclimated from seawater (SW; 38 ppt, salinity; 1291 mOsmol/l) down to dilute seawater (DSW; 20 ppt, salinity; 679 mOsmol/l). During acclimation to DSW haemolymph was only transiently hypoosmotic, becoming isosmotic to the medium over a 24-h period of acclimation. Na,K-ATPase specific activities in homogenates of the trichobranchiate gills from SW- and DSW-acclimated spiny lobsters were in the range of 2-3 μmol Pi/h/mg protein and were not significantly different. It has also been confirmed for the marine stenohaline crustaceans Maja crispata and Dromia personata that gill Na,K-ATPase maintains the same level of specific activity in SW- and DSW-acclimated crabs. The saponin-treated fraction of Na,K-ATPase activity in trichobranchiate gills was 67-89% and epipodites 63-64% over the native homogenates' activity and no differences in enzyme activities upon saponin treatment between SW- and DSW-acclimated spiny lobsters were found. Recovery of 6% and enrichment factor (1.6) of Na,K-ATPase in partially purified plasma membrane fractions of epipodites was relatively low and not different in SW- and DSW-acclimated spiny lobsters. In the hemiepipodite, negative short-circuit current was in the range from -16.7 to -22.7 μA cm(-2) and conductance varied in the range of 205-290 mS cm(-2), values which were not significantly different in spiny lobsters residing in SW or DSW. Very high conductance suggests leakiness of the hemiepipodite epithelium-cuticular complex. In contrast to the group of euryhaline hyperosmoregulating Crustacea in which activation of the specific activity of Na,K-ATPase upon acclimation to dilute seawater occurs, in marine osmoconformers there is no activation of the enzyme in dilute seawater. Based on the literature data and our own results, we have reported a correlation coefficient of 0.65 between specific activity of Na,K-ATPase and the sodium gradient (mmol Na/l; haemolymph-seawater ) between 12 species of osmoconforming and osmoregulating Crustacea. During evolution, hyperosmoregulating Crustacea have achieved internal osmolyte gradients generated by Na,K-ATPase and lowering the gill surface permeability. However these adaptive characteristics are not present in marine osmoconforming Crustacea, restraining them to migrate in the brackish water habitats.     

Prospects of using marine actinobacteria as probiotics in aquaculture

In the present study, optimum culture conditions for the production of extracellular polysaccharides (EPS) in submerged culture of an edible mushroom, Laetiporus sulphureus var. miniatus and their stimulatory effects on insulinoma cell (RINm5F) proliferation and insulin secretion were investigated. The maximum mycelial growth (4.1 g l⁻¹) and EPS production (0.6 g l⁻¹) in submerged flask culture were achieved in a medium containing 30 g l⁻¹ maltose, 2 g l⁻¹ soy peptone, and 2 mM MnSO₄·5H₂O at an initial pH 2.0 and temperature 25°C. In the stirred-tank fermenter under optimized medium, the concentrations of mycelial biomass and EPS reached a maximum level of 8.1 and 3.9 g l⁻¹, respectively. Interestingly, supplementation of deep sea water (DSW) into the culture medium significantly increased both mycelial biomass and EPS production by 4- and 6.7-fold at 70% (v/v) DSW medium, respectively. The EPS were proved to be glucose-rich polysaccharides and were able to increase proliferation and insulin secretary function of rat insulinoma RINm5F cells, in a dose-dependent manner. In addition, EPS also strikingly reduced the streptozotocin-induced apoptosis in RINm5F cells indicating the mode of the cytoprotective role of EPS on RINm5F cells.     

Branchial chloride cells in sea bass (Dicentrarchus labrax) adapted to fresh water,seawater, and doubly concentrated seawater

Branchial chloride cells (CC) were studied in sea bass (Dicentrarchus labrax) maintained in seawater (SW: 35 per thousand) or gradually adapted to and subsequently maintained in fresh water (0.2 per thousand) or doubly concentrated seawater (DSW: 70 per thousand). Changes were observed in the location, number, and structure of CCs, that were discriminated by light, scanning, and transmission electron microscopy, as well as by immunofluorescence on the basis of their high Na(+)/K(+)-ATPase antigen content. The number of CCs increased in both fresh water and doubly concentrated seawater compared to control fish maintained in SW. In both experimental conditions, these cells were found on the gill filament (as in control fish) and even on the lamellae, especially in hypersaline conditions. Structural changes concerned the shapes and sizes of CCs and their apical outcrops and particularly the structures of their functional complexes (mitochondria, tubular system, and endoplasmic reticulum), which developed significantly in DSW adapted fish. The changes in the expression of the Na(+)/K(+)-ATPase were evaluated by assessing the enzyme's density at the ultrastructural level following immunogold labeling. This parameter was significantly higher in doubly concentrated seawater. The adaptative significance of the quantitative and morphofunctional changes in branchial chloride cells is discussed in relation to the original osmoregulatory strategy of this marine euryhaline teleost.     

Growth and photosynthetic responses of Ulva lactuca (Ulvales,Chlorophyta) germlings to different pH levels

In ocean ecosystems, fluctuations in seawater pH affect CO₂ fluxes, fundamentally influencing the metabolism of marine algae, especially during the early stages of macroalgal development. In this study, short-term exposure tests (minutes) and prolonged culture experiments (eight days) were performed at different pH levels to investigate the growth and photosynthetic responses of Ulva lactuca (Ulvales, Chlorophyta) germlings. Both acidified and alkalized seawater significantly depressed algal photosynthesis during short-term exposure tests. Prolonged culture in acidified or alkalized seawater also notably decreased photosynthesis rates and growth rates of U. lactuca germlings, but increased energy consumption and lipid peroxidation, indicating damage to the germlings. Our results suggested that both lowered and increased pH levels of seawater exert significant physiological stress on U. lactuca germlings.