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In the production of natural sausage casings, three layers of serosa, muscular, and mucosa are removed from gastro-intestinal wall of sheep as waste materials. The submocusa layer is taken for further processing. There is no report about generating added value out of these wastes. In this study, a novel approach was introduced for bioconversion of waste gastro-intestinal wall (WGW) to a value-added product. Alkaline hydrolysis of WGW was investigated and the hydrolysate was utilized for cultivation of Dunaliella salina, a value-added biomass. The hydrolysate that contained the highest total soluble protein was used for three sets of cultivations on different medium compositions, i.e., (1) cultivations on the modified Johnson’s medium enriched with different percentage of hydrolysate (0.5, 1, 2.5, 5, and 10 (%v/v)), (2) cultivations on modified Johnson’s medium which was free of nitrogen and carbon sources and enriched with different percentage of hydrolysate (0.5, 1, 2.5, 5, and 10 (%v/v), and (3) cultivation on modified Johnson’s medium which was free of nitrogen source and enriched with 2.5% hydrolysate. The results showed that WGW contained 60.7, 8.4, 15.8, and 15.2% protein, lipid, moisture, and ash, respectively and the enrichment of the medium with the hydrolysate (2.5%) increased biomass productivity by 20%. Additionally, substitution of 2.5% hydrolysate for nitrogen source (KNO3) resulted in the same biomass productivity. The results of this study revealed the potential of the hydrolysate as an alternative for KNO3 in cultivation of D. salina. Overall, this work proposed a novel approach for converting waste gastro-intestinal wall to value.

Dunaliella salina is a unicellular microalga with the potential of industrial applications. Commercial-scale cultivation of D. salina requires extensive efforts on cost reduction. This can be achieved by minimization of nutrient cost and maximization of specific growth rate and productivity. To this end, in this study, mixotrophic cultivation of D. salina on crude glycerol (by-product of calcinated-fatty acid (CaFA) production process) was investigated in shake flasks and the influences on protein, carotenoid and chlorophyll contents of the cells were studied. The highest maximum specific growth rate and biomass productivity was achieved by cultivation on 2.5 g/L crude glycerol. The protein content was more than two folds and the carotenoid content was more than 60% higher than that of autotrophic cultivation. Subsequently, continuous cultivation of D. salina on crude glycerol in a helical photobioreactor was performed at the dilution rate of 0.52/d. The biomass productivity of 2.3 × 106 cell/(mL d) was achieved which was more than 75% of the values for previously reported autotrophic cultivations. Conclusively, continuous cultivation of D. salina on by-product of CaFA production process could be a potential process technology for production of D. Salina.

The effects of autotrophic, heterotrophic, and mixotrophic conditions were examined on micro green alga Dunaliella salina (UTEX 200) in terms of cell growth, total chlorophyll concentration, and lipid production. Results revealed a minimum growth rate of microalgae in the heterotrophic condition which is not recommended for D. salina. When the cells were cultured in mixotrophic conditions containing different concentrations of acetate or glucose, in 100 mM acetate or 60 mM glucose, the highest levels of growth rate and lipid production were obtained compared with autotrophic culture; however, the chlorophyll concentration decreased. Moreover, a significant correlation between ROS production and lipid synthesis was observed. When the combined acetate and glucose was added to the culture, no increase in growth rate and lipid synthesis was observed. This might be due to substrate inhibition. In the presence of glucose, the acidification of culture and bleaching occurred. Results indicated that lipid production is not affected by acidified conditions. In general, our results confirmed that mixotrophic culture with 100 mM acetate or 60 mM glucose is the most appropriate culture for D. salina concerning high lipid production as well as high growth rate in the shortest period of time.

The development of nanotechnology and the upsurge of interest in titanium dioxide (TiO2) nanoparticles, especially the anatase and rutile crystalline phases, in consumer products such as paint and sunscreen, has polluted the aquatic environment and had adverse effects on living organisms, especially algae. Microalgae help to preserve the aquatic ecosystem. Accordingly, the present study investigated the effects of anatase and rutile TiO2 nanoparticles on the growth, photosynthetic pigment (chlorophyll), photosynthesis, and respiration rate of two algae species, Dunaliella salina (at NaCl concentrations of 1.5 and 0.5 M) and Dunaliella tertiolecta (at NaCl concentrations of 0.5 and 0.17 M). Treatment with 50, 100, 150, and 200 ppm of TiO2 and nano-TiO2 revealed that nano-TiO2 inhibited the growth and decreased the specific growth rate, chlorophyll, and photosynthesis of both algal species. The rate of decrease was significantly lower at higher concentrations of NaCl in both species; however, the greatest significant difference was observed at lower concentrations of NaCl in the anatase phase. The respiration rate increased for 2 weeks but, especially at lower concentrations of NaCl, the rate of increase declined at higher concentrations after exposure to both substances, especially in the anatase phase. The findings reveal that nano-TiO2 has a toxic effect on Dunaliella algae and its effect depends on the concentration of NaCl. The toxic effect was shown to decrease at higher concentrations of NaCl.

Plastid terminal oxidase (PTOX) is a plastid-localized protein that acts as plastoquinol oxidase. The objective of this study was to determine the role of plastid terminal oxidase in response to drought stress and examine the differential response of PTOX pathway in both cultivated (Yousef and Morocco) and tolerant wild barley (spantaneum; HS) genotypes. Plants were subjected to different levels of soil water availability including control, mild and severe drought stress. Decrease in leaf relative water content as a result of progressive drought stressed led to decrease in stomatal conductance and increased enzymatic antioxidant machinery in all genotypes. Drought stress did not affect the PTOX transcript and protein level in cultivated genotypes Yousef and Morocco. However, PTOX gene expression and protein content significantly increased in HS genotype under severe drought. Assessment of PTOX activity via fast fluorescence induction curve in presence of PTOX specific inhibitor revealed that PTOX could efficiently oxidize plastoquinon pool and acts as alternative electron pathway in HS genotype. Results indicated that the role of PTOX in stress response was more significant in the wild barley than the cultivated barley varieties, where the emphasis has been on the desirable agronomic traits.

Heat shock proteins (HSPs) have vital roles during plant adaptation to biotic and abiotic stresses, as well as stress-free conditions. In the present study, we used a heterologous strategy of virus induced gene silencing to investigate the role of different classes of HSPs in Nicotiana benthamiana. Different growth and physiological parameters in silenced plants were evaluated under both control and salt stress conditions. Among the treatments, silencing of HSP70, especially under salinity regime, was found to have stronger impacts on growth, protein concentration, the accumulation of photosynthetic pigments, proline and total soluble carbohydrates content, malondialdehyde, the activity of antioxidant enzymes, performance index, relative water content and the ratio of K+/Na+, suggesting a more prominent role for HSP70 in both stressful and stress-free life cycle of the plants. Principal component analysis and hierarchical clustering indicated that HSP70 gene was silenced, the plants might effectively respond to stress by a higher accumulation of compatible solutes, like proline to protect the cell membranes and proteins from damage.

To study the effects of progressive drought stress on photosystem II behavior of wild type (Spantaneum) and cultivated barley (Morocco), different levels of soil water availability, including control, moderate, mild, and severe water stress (70%, 50%, 30%, and 10% water holding capacity of soil, respectively) and rehydration were used. Polyphasic OJIP fluorescence transient of Morocco plants exhibited a considerable increase in fluorescence intensity at O, J, and I steps under mild and severe stress relative to slight increase in wild barley. Values of fluorescence parameters and quantum efficiencies, including minimal fluorescence, relative variable fluorescence at phase J and I, maximal quantum yield of photosystem II (PSII), performance index, electron transport yield, and excitation transfer efficiency were influenced by water stress in both genotypes. These parameters were significantly less affected in wild type barley by progressive drought stress compared to Morocco. After re-watering, bot...

Effects of n-propyl gallate, a plastid terminal oxidase inhibitor involved in chlororespiration, on photosystem II efficiency in Dunaliella bardawil under low or high illumination was investigated. Rapid chlorophyll a fluorescence transients were recorded and analyzed according to JIP-test, which can quantify the photosystem II performance. The fluorescence transients O-J-I-P drastically decreased and almost reached a plateau when low light-grown cells were exposed for 96 h to 1, 2 and 4 mM n-propyl gallate. Very similar reductions in the efficiency of quantum yield of primary photochemistry (Φpo), the quantum yield for electron transport (ΦEo) and the inferred water-splitting complex activity (Fv/Fo) were found in the same inhibitor concentrations. However, no statistically significant change in fluorescence intensity and photosystem II efficiency was found when algal cells were exposed to the inhibitor concentrations up to 2 mM under high light intensity. The results indicated that inhibitory effects of n-propyl gallate on photosystem II electron flow in D. bardawil cells are dependent on environmental conditions. It is also demonstrated that n-propyl gallate is a multi-target inhibitor of growth kinetics as well as photosynthesis. In addition, we found that the donor side of photosystem II acts as main target place of the inhibitor.

In this work, we investigated the importance of phytoene desaturase (PDS) during salt stress in plants. For this aim, we transiently suppressed the corresponding gene of Nicotiana benthamiana using a Tobacco Rattle Virus silencing system. After salinity stress, the silenced plants were assayed for different growth and physiological parameters. The silenced plants were found to be induced more stunted and feeble shoots and roots than the negative controls. The results indicated that the concurrent incidence of salinity stress and viral infection did not have an additive influences on the plant growth and physiological parameters. Importantly, our results showed a more detrimental influence of salinity on plant growth and physiology after PDS silencing. Compared to the control, salt-stressed plants contained more proline, and malondialdehyde, less amount of photosynthetic pigments, higher Na+/K+ ratios in shoots and roots and lower relative water content after PDS silencing. The activity of antioxidant enzymes were also decreased in the plants coped with salinity or Tobacco Rattle Virus. It was also represented that simultaneous silencing of the PDS gene and salinity treatment resulted in a significant decrease of the performance index (PIABS) and an increase of dissipation per active reaction center (DIo/RC) reflecting severe injuries in photosynthetic machinery under such a condition. Overall results suggested that PDS silencing could increase the sensitivity of plants to salinity.

The ability of two Dunaliella salina strains (AS-B1 and IR-1) and Dunaliella bardawil (UTEX-2538) in the sedimentation of different concentrations (0, 500, 1000, 2000, 3000 and 4000 μM) of aluminum were studied for duration 120 min. In these strains, increase in aluminum concentration was concomitant with the reduction of cell number in supernatant as the index of the sediment aluminum by algal cells-Al 3+ ions binding; cell agglutination and sedimentation caused by presence of aluminum. The ability of D. bardawil was higher in sedimentation of aluminum. The results of adding EDTA to cell suspensions for binding the aluminum confirmed the role of binding algal cells to aluminum in cell agglutination and sedimentation and consequently aluminum bioremediation. In addition, the results imply that the cell agglutination and sedimentation in D. bardawil is higher than Dunaliella salina . It seems this strain has a greater capability for sedimentation of aluminum and can utilize aluminum from aquatic environments for bioremediation.

Drought stress is one of the most important abiotic factors which adversely affect growth, metabolism and yield of crops worldwide. The objective of this study was to determine the effects of drought stress on photosynthesis in barley and examine the differential responses of photosynthetic apparatus in relatively tolerant (Yousof) and susceptible (Morocco) barley genotypes. Plants were subjected to different levels of soil water availability including control, mild and severe drought stress. In both genotypes, drought stress led to decrease in chlorophylls, β-carotene and stomatal conductance accompanied by decrease in CO2 assimilation rate. Significant increase in α-tochoperol content was only observed in Yousof cultivar under drought stress. Initial slope and plateau phase of CO2 response curve of drought-stressed plants as well as polyphasic chlorophyll a fluorescence transient curve (OJIP test) and fast fluorescence induction kinetics were influenced by drought stress. These parameters were more affected in Morocco cultivar by drought stress compare with Yousof. Drought stress also resulted in reduction of D1 protein content in both genotypes and accelerated photoinhibition process. Based on our results, stomatal conductance is the main factor limiting photosynthesis in Yousof genotype under mild drought stress. However, in Morocco, in addition to stomatal limitation, damage to photosystem II (PSII), reduced electron transport and carboxylation efficiencies were important parts of limitation in photosynthesis. Severe drought stress resulted in structural and biochemical impairment of light-dependent reactions as well as carboxylation process of photosynthesis in both genotypes. Alpha-tocopherol showed an important protective role against drought stress in Yousof cultivar as a relatively drought-tolerant cultivar.

Electron transport in photosystem II (PSII) and photosystem I (PSI) was estimated in terms of chlorophyll fluorescence and changes in P700 redox, respectively, in the unicellular green alga Dunaliella salina in the presence or absence of a nitrogen source in the culture medium. In a nitrogen-containing medium, the quantum yield of PSII (Φ(II)) and that in PSI (Φ(I)) were at the same level in low light, but cyclic electron transport around photosystem I (CET-PSI) was induced under high light as estimated from an increase in Φ(I)/Φ(II). High light might further enhance the rate of electron transport in PSI by inducing the state 2 transition, in which the distribution of light energy is shifted to PSI at the expense of PSII. Nitrogen deficiency resulted in a decrease in Φ(II) and an increase in Φ(I). As a consequence, the rate of CET-PSI was expected to increase. The high CET-PSI under N deficiency was probably associated with a high level of energy quenching (qE) formation in PSII.

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.

A highly promising procedure to obtain seedlings of Taxus baccata L. has been developed, which involves a combination of in vitro embryo culture and growth under hydroponic conditions. Embryos isolated from freshly collected seeds were 100% sterile, even though the seeds were not treated with acid or soaked in water prior to culturing. The embryo germination level of non-leached seeds was slightly lower (85%) than those leached in running water for 7 d (100%). The leached embryos germinated with extended roots while the non-leached embryos had abnormal shapes. The embryos cultured on media supplemented with an absorbent (PVP or activated charcoal) had extended roots and shoots and were a larger size without any browning, as compared to those grown without the supplement; activated charcoal gave better results. There were no significant differences in germination rates of T. baccata embryos between the media with differing strengths of macronutrients; however, for further development of the shoot, it was necessary to sub-culture the seedlings in MS in the light. To obtain seedlings with longer roots, they had to be maintained in one-half strength MS in darkness. Approximately 90% of the plants survived when grown hydroponically for 2 mo. The surviving plants showed well-extended roots and were a good starting material for genomic, proteomic, and conservational studies as well as Taxol permeabilization investigations.

Dunaliella species accumulate carotenoids and their role in protection against photooxidative stress has been investigated extensively. By contrast, the role of other antioxidants in this alga, has received less attention. Therefore, the components of the ascorbate-glutathione cycle, along with superoxide dismutase (E.C. 1.15.1.1) and peroxidase (E.C. 1.11.1.11) activity were compared in two strains of Dunaliella salina. Strain IR-1 had two-fold higher chlorophyll and beta-carotene concentration than Gh-U. IR-1 had around four-fold higher superoxide dismutase, ascorbate peroxidase and pyrogallol peroxidase activities than Gh-U on a protein basis. Ascorbate and glutathione concentrations and redox state did not differ between strains and there was little difference in the activity of ascorbate-glutathione cycle enzymes (monodehydroascorbate reductase [E.C. 1.6.5.4], dehydroascorbate reductase [E.C. 1.8.5.1] and glutathione reductase [E.C. 1.8.1.7]). The response of these antioxidants to high light and low temperature was assessed by transferring cells from normal growth conditions (28 degrees C, photon flux density of 100 micromol m(-2) s(-1))to 28 degrees C/1200 micromol m(-2) s(-1); 13 degrees C/100 micromol m(-2) s(-1); 13 degrees C/1200 micromol m(-2) s(-1) and 28 degrees C/100 micromol m(-2) s(-1) for 24 h. Low temperature and combined high light-low temperature decreased chlorophyll and beta-carotene in both strains indicating that these treatments cause photooxidative stress. High light, low temperature and combined high light-low temperature treatments increased the total ascorbate pool by 10-50% and the total glutathione pool by 20-100% with no consistent effect on their redox state. Activities of ascorbate-glutathione cycle enzymes were not greatly affected but all the treatments increased superoxide dismutase activity. It is concluded that D. salina can partially adjust to photooxidative conditions by increasing superoxide dismutase activity, ascorbate and glutathione.

In this study, carotenoid and glycerol production in two unicellular green algae (Dunaliella salina and D. viridis) isolated from the Gave-Khooni salt marsh grown in media containing five different salt concentrations (0.17, 1, 2, 3, and 4 M NaCl) were evaluated under sterile conditions. Algae growth decreased as the medium salinity increased. Optimum growth of D. salina and D. viridis were obtained at 2 and 1 M NaCl, respectively. As salinity increased, glycerol and carotenoid production were increased in D. salina, whereas lower values for these products were produced in D. viridis under the same conditions. Furthermore, the cell color of D. salina changed from green to orange-red following accumulation of carotenoid, but the color of D. viridis was not changed. Thereby, it seems that the Iranian D. salina may be suitable for carotenoid production (betacarotene) on a large scale. In addition, since carotenoid compounds enhance the efficiency of photosynthesis and glycerol synthesis, it appears that the pathway for glycerol production and mechanisms of salt tolerance in D. viridis are unique from those of D. salina.

Three strains of Dunaliella salina (I, G and A) were cultivated under the climatic conditions of Iran, in open ponds to compare the β-carotene production and the specific rate of growth. The experiments were accomplished in two separate stages. In the first stage, the cells were grown in ponds on nutrient-rich medium containing 2 M NaCl to obtain the necessary biomass. In the second stage, cells were stressed on nutrient-poor medium containing 2.5 M NaCl for β-carotene induction. The results showed that the specific growth rate of strain I was the highest during the first stage, whereas during the second stage, the growth rates of three strains were approximately the same. The overall results indicated that strain G had the highest potential for β-carotene accumulation of the strains tested and hence it was concluded that this strain is more suitable for outdoor cultivation under the climatic conditions of Iran than the other two.

We studied the effects of n-propyl gallate, which is a plastid terminal oxidase inhibitor involved in chlororespiration, on photosynthesis and physiological parameters in Dunaliella salina grown under different salinities and under low or high irradiance using chlorophyll a fluorescence transient measurements and pigment analysis. The inhibitor up to 1 mM had an additive significant effect on the photosynthetic efficiency in the cell suspensions grown under low salinity and irradiance. However, in the presence of high n-propyl gallate concentration (4 mM), there was a negative effect on all physiological aspects. In contrast, this high concentration of the inhibitor could enhance efficiency of electron transport and growth parameters under high irradiance. On the other hand, with salinity increase, the unfavorable effects of high inhibitor concentration on the efficiency of photosystem II were less evident than of low salinity. Interestingly, n-propyl gallate high concentration had a positive effect on fluorescence and on physiological parameters when high salinities-grown cells were exposed to high irradiance. The results suggest that there is a rational correlation between increase of salinity and algae ability to bypass n-propyl gallate inhibited plastid terminal oxidase function and also direct influence of its lethal concentration on photosystem II compartment. The ability is especially substantial when the increase of salinity is accompanying high irradiance. Furthermore, these data show that algal responses to inhibitor concentrations are different under various environmental conditions.

Dunaliella is a halotolerant green alga that now belongs to the phylum Chlorophyta and family Polyblepharidaceae (Avron and Ben-Amotz 1992; Garcia et al., 2007). It lacks a rigid cell wall nevertheless it can grow in aquatic environments varied salinities from 0. 5 to 5.0 M NaCl (Shariati & Hadi, 2000, Phadwal & Singh, 2003; Jahnke & White, 2003). Under stress conditions, Dunaliella species can accumulate significant amounts of valuable chemical matters such as carotenoids (Hosseini Tafreshi & Shariati, 2006; Hadi et al., 2008), glycerol (Hadi et al., 2008), vitamins and proteins (Ghoshal et al., 2002). The mechanism by which Dunaliella cells can adapt to this wide range of salt concentrations was shown to be based on the ability of the alga to change its intracellular concentration of glycerol (Raja et al., 2007). In fact, the accumulation of glycerol in this alga is regulated by external water activity rather than the specific solute effect (Shariati & Lilley, 1994). In this condition, glycerol acts as a ‘compatible solute’ that protects enzymes against both inactivation and inhibition (Telfer, 2002). It was also shown that both the glycerol synthesis under hypertonic conditions and its elimination under hypotonic condition are independent of protein synthesis and occur in the light or dark (Shariati & Lilley, 1994). Therefore recently, algal biotechnology has made major advances, and microalgae like Dunaliella sp. are cultivated for the production of carotenoids and glycerol (Hosseini Tafreshi & Shariati, 2009; Spolaore et al., 2006). The potential ability of carotenoids to act as antioxidants and immunomodulatory agents has led to more active research investigating their application in the prevention of human cancers (Chidambara Murthy et al. 2005). Also, this alga accumulates large amounts of β-carotene (up to 14% of dry weight) in conditions such as high light intensity (Coesel et al., 2008), increased temperature (Ben-Amotz, 1996; Gomez & Gonalez, 2005), high salinity (Hadi et al., 2008) and nutrient deficiency (Marin et al., 1998) such as sulfate deficiency (Aghaii & Shariati, 2007) and nitrate deficiency (Shariati & Zoofan, 2003). Dunaliella β-carotene is used in the food (Dufosse et al., 2005), cosmetic, and pharmaceutical industries as a colorant, antioxidant (Chidambara Murthy et al., 2005), anti-tumor agent, and heart disease preventive (Tornwall et al., 2004), in addition to its characteristic as precursor of vitamin A. Among the parameters that can significantly influence the production of biomass and βcarotene in open ponds is the selection of the area (Hosseini Tafreshi & Shariati, 2006). Dunaliella production plants are located in areas having a hot and dry climate with minimal

The microalga Dunaliella salina is the best commercial source of natural beta-carotene. Additionally, different species of Dunaliella can accumulate significant amounts of valuable fine chemicals such as carotenoids, glycerol, lipids, vitamins, minerals and proteins. They also have a large potential for biotechnological processes such as expressing of foreign proteins and treatment of wastewater. In this review, we discussed several biotechnological aspects of the mass cultivation of D. salina like strain selection, carotenoid induction, culture conditions, culture systems and downstream processes. We also discuss several traditional and new applications of the genus.

The aim of this research is to analyze the two varieties of British and American English in respect of their phonetic differences. Although the language of Britain and America is English, there are some pronunciation, vocabulary, spelling, grammatical, idiomatic and other differences between them. In this research, the phonetic differences between these two English varieties have been analyzed in terms of their vowels (including monophthongs and diphthongs) and consonants. The method used in this research is comparative whose data have been collected through library and internet sources. Comparative method is a linguistic method used to compare and contrast two or more languages. The obtained results, based on the comparison between British and American pronunciation, will get English language learners to the recognition, conscious knowledge and correct usage of British and American English.