Native Algae Research Articles

Bio-removal of Zn from contaminated water by using green algae isolates

Abstract Wastewater is one of the main sources of the heavy metals. These hazardous compounds find their way through water resources and consequently threaten human being’s life and other living organisms. Several solutions have been proposed to address this issue. Nowadays, using algae to eliminate these elements is the most effective method in terms of cost and time in comparison with other biological and chemical methods. This research was carried out to evaluate the capability of native algae species in Zn removal from industrial sewage. A split plot (in time) experiment which consists of one main factor (time) and three subsidiary factors including basic solution at two levels, algae isolates at three levels and Zn concentration at three levels of 0 (blank solution), 10, and 100 mg/L from ZnSO4.7H2O) was conducted. Firstly, the dominant and fast growing algae isolates were separated from the depth of 20 cm, in Botanical Garden of Tabriz University and Khalatpoushan Research Station, Tabriz, Iran. Then, three efficient isolates were selected for the second phase of the experiment. After sufficient growth of the superior selected algae isolates on minimal medium, algal suspension was prepared and the population adjusted to 3×104 cell per mL, and then the suspension was transferred to the Erlenmeyer flasks (100 mL) including Tabriz wastewater and basic solution. OD, pH and Zn concentration was measured in intervals of 3, 6, 9, 15 and 20 days. Based on the results, the highest Zn absorption by two algae isolates was recorded in the minimum medium with the blank solution and in the municipal wastewater with the concentrations of 10 and 100 mg/L which can be concluded that both isolates were capable of eliminating contamination effectively in the natural environment compared to the laboratory condition. At the concentrations of 0 and 10 mg/L, almost all Zn was removed from both medium and at the concentrations of 100 mg/L, approximately 90% of Zn was removed from both culture mediums. Accordingly, it can be concluded that both algal isolates can be considered as a suitable bio-remover for removing Zn form polluted waters.

Effectiveness of elimination inflowing algae in water treatment plant using natural algae remover

Effectiveness of elimination inflowing algae in water treatment plant using natural algae remover

Present-Day Distribution and Potential Spread of the Invasive Green Alga Avrainvillea amadelpha Around the Main Hawaiian Islands

Algal assemblages are critical components of marine ecosystems from the intertidal to mesophotic depths; they act as primary producers, nutrient cyclers, and substrate providers. Coral reef ecosystems can be disrupted by stressors such as storm events, effluent inundation, sudden temperature shifts, and non-native invaders. Avrainvillea amadelpha is an invasive green alga that was first recorded in the main Hawaiian Islands on the west shore of Oahu and has continued to be of concern due to its extreme competitiveness with native algae and seagrasses. It has spread rapidly across the island of Oahu, decreasing the biodiversity of the benthos from shorelines to ~90 m depth. We employed a boosted regression tree modeling framework to identify highly vulnerable regions prone to invasion. Our model indicated that regions exposed to minimal bottom currents and at least five degree heating weeks are particularly susceptible to A. amadelpha colonization. Additionally, we extrapolated our model to the main Hawaiian Islands and forecasted how a 25% increase in statewide annual maximum degree heating weeks may change habitat suitability for A. amadelpha. Across all islands, we identified particularly vulnerable “hotspot” regions of concern for resource managers and conservationists. This manuscript demonstrates the utility of this approach for identifying priority regions for invasive species management in the face of a changing climate.

Use of standard test organisms for sound validation of UV-based ballast water treatment systems

To challenge ballast water treatment system (BWTS) efficacy for organisms in the size-class 10–50 μm, intake concentration during tests must reach certain minimum requirements. Often, natural concentrations are too low to meet intake requirements and standard test organisms (STOs) are added. We tested the robustness of Tetraselmis suecica and Odontella sp. to a range of UV-treatments to explore fluences needed to meet the IMO discharge standard (<10 org. ml−1) evaluated using two viability assessment methods. To meet discharge standards, fluences of >1000 mJ cm−2 were required using vital stain whereas 135–500 mJ cm−2 were needed using regrowth assays. Besides, results suggest that T. suecica and Odontella sp. were at least as robust as natural algae towards UV-treatments. We suggest the advantageous use of these species as STOs in test water to support intake water requirements and to obtain more conservative validation of UV-based BWTS to ensure more environmental protective procedures.

Development of a novel automated analytical method for viability assessment of phytoplankton used for validation of ballast water treatment systems

To limit the spreading of aquatic invasive species, regulations require ships’ ballast water to be treated before discharge. To validate ballast water treatment system (BWTS) performance, treated water is analyzed for living organisms in different size classes. Quantitative assessment of the size class 10–50 μm (mainly phytoplankton) is carried out using the vital stain method, which requires labor-intensive manual microscope counts of fluorescent (i.e., living) cells. The method is slow, demands specialized personnel, and is challenged by subjectivity and mobile organisms. Using a high-content screening platform (HCS-Platform) and image analysis, we developed an automated, objective and faster quantification method. The automated method neutralized subjectivity by using fixed cell recognition parameters for image analysis. The implementation of membrane filters gently manipulated the organisms into a 2D plane that reduced mobility. Quantifications were performed at different concentrations using monocultures of slow-moving Rhodomonas salina, highly mobile Tetraselmis suecica and natural algae. Results were compared to the standard manual counting procedure. Automated counts of monocultures were comparable to manual counts at low and medium concentration levels. Manual counts of T. suecica at high concentration levels were significantly lower compared to automated counts stressing the challenge to count mobile cells in 3D. Natural algal counts were similar for both counting approaches, but accuracy was challenged by colony forming species and high number of algal species ~ 10 μm. Automated counts were significantly faster than manual counts. In conclusion, the HCS-Platform showed promising results as an alternative quantitative phytoplankton assessment method for BWTS validation.

Unexpected reproductive traits of Grateloupia turuturu revealed by its resistance to bleach-based biosecurity protocols

Abstract A non-indigenous alga, Grateloupia turuturu (Halymeniales, Rhodophyta), was discovered in the Damariscotta Estuary (Maine, USA) in 2017, over 200 km north of its last reported location. Because of the presence of coastal facilities (aquaculture, marine laboratories) among potential vectors, we evaluated a mandated biosecurity protocol, namely, seawater treated with bleach at 50 ppm (50 mg l−1) free chlorine for ≥2 h and measured using test strips, for lethality against G. turuturu and several native algae. We report unexpected resilience to bleach-treatment (Mastocarpus &gt; Grateloupia &gt; Palmaria). Holdfasts of G. turuturu and Mastocarpus stellatus survived 50 and 1000 ppm free chlorine, respectively. Cystocarps on cultured blades of G. turuturu were particularly resistant to bleach-treatment; they produced outgrowths on fragmenting blades that became fertile tetrasporophytes in culture, suggesting reproductive adaptations of cystocarps to stressful conditions that may have broader evolutionary significance. Juvenile, microscopic crusts of G. turuturu were also bleach-resistant if covered by diatoms, and developed upright axes. Free chlorine test strips were inaccurate in seawater, and their use could increase the risk of failed biosecurity as a vector for invasion. Because chlorination at economic and environmentally safe levels is insufficient, we recommend a combination of treatments for comprehensive biosecurity.

Degradation of Eight Sulfated Polysaccharides Extracted from Red and Brown Algae and Its Impact on Structure and Pharmacological Activities.

Degradation represents a strategy to improve the biopharmaceutical properties of native algae sulfated polysaccharides (SP) with high Mw. The aim of this study was to compare the degradability of four sulfated xylogalactans (SXG) and four fucose-rich sulfated polysaccharides (FRSP) extracted from red and brown algae, respectively, using three simple methods causing no desulfation as well as to examine the chemical and pharmacological changes of the resulting fractions. The achieved degradation proved to be dependent on the basic glycan structure of the SP. Treatment with hydrogen peroxide (3%, 4 h, 50 °C) led to the most efficient degradation of both FRSP and SXG. The Mw decrease was associated with distinct reduction of the activities (complement inhibition (>) elastase inhibition > C1-INH potentiation) and resulted in a modified pharmacological profile. Despite their much lower degree of sulfation, some of the fractions with Mw < 15 kDa exhibited similar or even stronger activities than heparins, whereas they had only weak anticoagulant effects.

Microalgae–bacteria consortium treatment technology for municipal wastewater management

The discharge of inadequately treated municipal wastewater in the developing countries is the major cause of environmental pollution in urban areas. However, the effort was made to minimize this burden using conventional wastewater treatment methods that require high capital and operational costs and are not affordable. Researchers are still looking for the cost-effective, efficient and environmentally compatible wastewater treatment technologies. Hence, this study aimed to investigate the potential of native microalgae–bacteria consortia for the removal of nutrient and organic pollutants from primary-treated municipal wastewater. Microalgae–bacteria treatment system was established by combining pre-cultured native algae consortia dominated by Chlorella sp., Chlamydomonas sp. and Scenedesmus sp. of the class Chlorophyceae with naturally existing municipal wastewater bacteria. Microalgae–bacteria culture acclimatization was performed and the actual experiment was carried out at 18% culture to wastewater by volume in photobioreactor at a light intensity of 120 μE/m2s. The maximum removal of TKN 69%, TP 59%, PO43−_ P 73%, COD 84% and BOD5 85% was observed in the combined treatment system, whereas for bacteria stand-alone treatment system, the maximum removal of TKN 31%, TP 56%, PO43−_ P 50%, COD 44% and BOD5 52% was recorded. Statistically significant differences were observed for the removal of NH3–N, TKN and PO43−_ P at p < 0.05 but statistically insignificant differences were observed for TP, COD and BOD5 in the combined treatment system. The study results suggest that the native microalgae consortia identified in the local environment can effectively reduce organic and nutrient pollutants from the primary-treated municipal wastewater. Generally, the performance evaluation of combined microalgae–bacteria treatment technology was efficient in municipal wastewater management and promising to scale up at industrial level.

Submarine Groundwater Discharge Differentially Modifies Photosynthesis, Growth, and Morphology for Two Contrasting Species of Gracilaria (Rhodophyta)

On many tropical reefs, submarine groundwater discharge (SGD) provides a substantial and often overlooked nutrient source to nearshore ecosystems, yet little is known about the impacts of SGD on the biology of reef organisms. To address this, the physiological responses of the endemic rhodophyte Gracilaria coronopifolia and an invasive congener, Gracilaria salicornia, were examined across an SGD gradient in the field and laboratory. Tissue samples of both species were cultured for 16 days along an onshore-offshore SGD gradient at Wailupe, Oahu. G. salicornia tolerated the extremely variable salinity, temperature, and nutrient levels associated with SGD. In marked contrast, half of G. coronopifolia plants suffered tissue loss and even death at SGD-rich locations in the field and in laboratory treatments simulating high SGD flux. Measurements of growth, photosynthesis, and branch development via two novel metrics indicated that the 27‰ simulated-SGD treatment provided optimal conditions for the apparently less tolerant G. coronopifolia in the laboratory. Benthic community analyses revealed that G. salicornia dominated the nearshore reef exposed to SGD compared with the offshore reef, which had a greater diversity of native algae. Ultimately, SGD inputs to coastal environments likely influence benthic community structure and zonation on otherwise oligotrophic reefs.

Progress in biosynthesis of phycobiliprotein

Photosynthesis has existed on the earth for about 4 billion years, and the photosynthetic organisms have emerged with the production of photosynthesis. In order to effectively capture light energy and adapt their living environment, algae and higher plants have evolved different light-harvesting antenna systems during the long evolution, one is the chlorophyll based light harvesting systems present mainly in terrestrial organisms, such as higher plants; and the other is the phycobiliprotein (PBP) based phycobilisome system mainly in aquatic organisms, such as cyanobacteria, red algae and cryptophytes. As a special light-harvesting pigment-protein complex, PBP can not only capture light energy, also shows many unique characteristics, such as good fluorescent properties and lots of pharmaceutical activities. Therefore, PBPs have been extensively studied and commercially used in foods, cosmetics, biotechnology detection, pharmacological and medicine fields. Due to its broad excitation spectrum, nontoxic, stable fluorescence and high quantum yield, PBPs have also employed as valuable fluorescent probes for immunological diagnosis. At present, there are mainly two ways to obtain fluorescent PBPs, one is to purify PBP directly from natural algae, and the other is to express recombinant PBP using genetic engineered host cells. Purification of PBPs from natural algae has many disadvantages, such as higher cost, complicated processes, and many by-products. Using genetically engineered cell biotechnology, large-scale and low-cost production of recombinant PBPs can be achieved, which can help solve problems such as source, quality control and so on. Since natural PBPs are mostly in the form of polymers such as trimers or hexamers, it is difficult to study the assembly process. Through genetic engineering techniques, monomers or subunits can be obtained, which will help to study the assembly process of PBP. In addition, novel recombinant PBPs can be produced by molecular design to improve their fluorescence and biological activity. The study of the biosynthesis will help to elucidate the relationship between the structure and spectral properties of PBPs and provide a basis for the construction of solar energy utilization systems. This review mainly focuses on the progress of combinational biosynthesis and in vivo reorganization of PBP in heterologous genetically engineered host cells, with emphasis on different genes coding for apo-PBPs and genes coding for different enzymes which catalyze the production of phycobilins from endogenous heme, along with the recent progress of three types of lyases which catalyze the covalently binding of phycobilins with apo-PBPs. The possible applications of these valuable recombinant PBPs were proposed as well.

UV fluences required for compliance with ballast water discharge standards using two approved methods for algal viability assessment

This study investigates the extra UV fluence needed to meet the International Maritime Organisation's ballast water discharge standards for the 10–50 μm size-class using the approved vital stain (VS) method compared to the Most Probable Number (MPN) method for organism viability assessment. Low- and medium pressure UV collimated beam treatments were applied to natural algae collected in temperate and tropical water environments and analysed using both methods. About 10 times higher UV fluence was required to meet discharge standards when using VS compared to MPN. Implementing a dark-hold period after UV treatments decreased algal viability. Length of dark-hold period to meet discharge standards decreased with increasing UV fluence. No significant differences between temperate and tropical samples were observed. The results showed that UV treated algae assessed using the VS method could meet discharge standards by increasing fluence and/or introducing a dark-hold period.

The Global Dispersal of the Non-Endemic Invasive Red Alga Gracilaria vermiculophylla in the Ecosystems of the Euro-Asia Coastal Waters Including the Wadden Sea Unesco World Heritage Coastal Area: Awful or Awesome?

Gracilaria vermiculophylla (Ohmi) Papenfu AŸ 1967 (Rhodophyta, Gracilariaceae) is a red alga and was originally described in Japan in 1956 as Gracilariopsis vermiculophylla. It is thought to be native and widespread throughout the Northwest Pacific Ocean. G. vermiculophylla is primarily used as a precursor for agar, which is widely used in the pharmaceutical and food industries. It has been introduced to the East Pacific, the West Atlantic and the East Atlantic, where it rapidly colonizes new environments. It is highly tolerant of stresses (nutrient, salinity, temperature) and can grow in an extremely wide variety of conditions; factors which contribute to its invasiveness. It invades estuarine areas where it out-competes native algae species and modifies environments. The following European coastal and brackish water seas are already invaded: Atlantic, North Sea, Mediterranean and Baltic Sea. The Euro-Asian brackish Black-Sea have not yet been invaded but are very vulnerable to intense invasion with G. vermiculophylla because they are isolated from direct marine influences and have a harsh environment with large salinity, nutrient and temperature fluctuations.

Domestication and Proliferation of Algae Cultures for Boosting Efficiency of Waste-water Treatment through Symbiosis

Native algae strains have been domesticated and stimulated in activated sludge wastewater treatment operations. The term of domestication indicates that we did not add any selected algae to the wastewater, but made use of species existing in the system. The term of proliferation indicates a stimulation of the biological oxidation process by provision of CO2 with the air stream and illumination of the reactor. The idea of domestication of algae present in communal wastewater systems was demonstrated. Stimulation of the system with domesticated algae community did improve efficiency of the treatment process. Removal of organic components in terms of reduction of chemical and biological oxidation demands (dissolved COD, BOD) as well as nitrogen and phosphorous contents was superior to extent of removal in conventional activated sludge system. We did conclude that conventional systems lack available light and carbon resources for these microorganisms. Upon providing these, symbiotic operation can contribute to reducing greenhouse gas emissions and increase of the rate of pollutants removal kinetics. Symbiotic operation increased the production of biomass expressed in terms of total suspended solids. Biodiesel potential of the filterable biomass was in the range of 8-18%. Because of technical difficulties in manipulation of the excess sludge other than biodiesel synthesis processing scheme has been recommended for economically viable processing.

Habitat associations of an expanding native alga

There are many examples of native macrophytes becoming locally dominant and spreading outside their traditional distributions, but the causes and impacts are often not understood. In New South Wales, Australia, the green alga Caulerpa filiformis is undergoing a range expansion and has transitioned from a subdominant to a dominant alga on several rocky shores around the Sydney coastline. Here we investigated relationships between established patches of C. filiformis, the habitat it occupies and associated algal communities at multiple subtidal sites over the green alga's 700 km range. We tested the following predictions: 1) C. filiformis cover differs among substrata, being greatest on turf-forming algae; 2) C. filiformis cover is positively related to environmental variables linked to increased sedimentation (e.g. reduced reef width, surface slope, increased rugosity and distance from shore); 3) occurrence of C. filiformis is associated with a change in macrophyte community structure and a reduction of macrophyte richness; 4) intact native algal canopies inhibit C. filiformis spread, but turf-forming algae and bare sand are susceptible to invasion. Substratum associations were highly consistent among sites, but contrary to our prediction, C. filiformis was most commonly associated with rock or rock + sand substratum and less frequently associated with turf-forming algae substratum. C. filiformis cover was negatively correlated with reef width, which explained most of the variation observed, although local scale variables distance from shore, reef slope, and water depth were also correlated with C. filiformis cover. Algal diversity and community composition typically differed in the presence of C. filiformis, often with a reduction of algal abundances, in particular Sargassum spp., although results varied among substrata and sites. However, monitoring of borders suggested that C. filiformis does not invade and outcompete undisturbed adjacent canopy-forming algae over a 12 month period. Our results suggest that disturbance processes (possibly linked to sedimentation) acting at the site and quadrat scale are likely important determinants of C. filiformis cover and spread, and hence its potential ecological impacts.

Effectiveness of a combined ethanol–aqueous extract of alga Cystoseira compressa for the quality enhancement of a chilled fatty fish species

Seaweeds have attracted an increasing attention as a new source for bioactive compounds, these include preservative compounds. This study is a first attempt to employ alga Cystoseira compressa for the preservation of chilled fish. For it, a combined ethanol–aqueous extract of this alga was included in the icing system and employed as chilling medium for the storage of horse mackerel (Trachurus trachurus) for 11 days. On the basis of the microbial groups (aerobes, psychrotrophs, proteolytic, lipolytic and Enterobacteriaceae bacteria) assessed, an inhibitory effect (p < 0.05) on microbial activity in horse mackerel muscle was observed as a result of including the alga extract in the icing medium; such preservative effect was also proved by chemical determinations related to microbial activity (pH and trimethylamine values). Additionally, a significant decrease (p < 0.05) of lipid hydrolysis (free fatty acids formation) and oxidation (fluorescent compounds formation) in fish was also observed as a result of the presence of C. compressa extracts in the icing medium. The icing medium proposed in this work (i.e., the combination of ethanolic and aqueous extracts of C. compressa) may constitute a promising strategy to the application of natural algae extracts for fatty fish storage and enhance quality retention during commercialisation.

Fluorescence Properties of Chlorella sp. Algae

Water quality and its fast and reliable monitoring is the challenge of the future. Design of appropriate biosensors that would be capable of non-invasive identification of water pollution is an important prerequisite for such challenge. Chlorophylls are pigments, naturally presented in all plants that absorb light. The main forms of chlorophyll in algae are chlorophyll a and chlorophyll b, other pigments include xantophylls and beta-carotenes. Our aim was to characterize endogenous fluorescence of the Chlorella sp. algae, present naturally in drinking water. We recorded spatial, spectral and lifetime fluorescence distribution in the native algae. We noted that the fluorescence was evenly distributed in the algae cytosol, but lacked in the nucleus and reached maximum at 680-690 nm. Fluorescence decay of chlorella sp. was double-exponential, and clearly shorter than that of its isolated pigments. For the first time, fluorescence lifetime image of the algae is presented. Study of the fluorescence properties of algae is aimed at the improvement of water supply contamination detection and cleaning.

Evaluation of phenotype stability and ecological risk of a genetically engineered alga in open pond production

Genetically engineered (GE) algae offer the promise of producing food, fuel, and other valuable products with reduced requirements for land and fresh water. While the gains in productivity measured in GE terrestrial crops are predicted to be mirrored in GE algae, the stability of phenotypes and ecological risks posed by GE algae in large-scale outdoor cultivation remain unknown. Here, we describe the first US Environmental Protection Agency (EPA)-sanctioned experiment aimed at understanding how GE algae perform in outdoor cultivation. Acutodesmus dimorphus was genetically engineered by the addition of two genes, one for enhanced fatty acid biosynthesis, and one for recombinant green fluorescence protein (GFP) expression; both the genes and their associated phenotypes were maintained during fifty days of outdoor cultivation. We also observed that while the GE algae dispersed from the cultivation ponds, colonization of the trap ponds by the GE strain declined rapidly with increasing distance from the source cultivation ponds. In contrast, many species of indigenous algae were found in every trap pond within a few days of starting the experiment. When inoculated in water from five local lakes, the GE algae's effect on biodiversity, species composition, and biomass of native algae was indiscernible from those of the wild-type (wt) progenitor algae, and neither the GE nor wt algae were able to outcompete native strains. We conclude that GE algae can be successfully cultivated outdoors while maintaining GE traits, and that for the specific GE algal strain tested here they did not outcompete or adversely impact native algae populations when grown in water taken from local lakes. This study provides an initial evaluation of GE algae in outdoor cultivation and a framework to evaluate GE algae risks associated with outdoor GE algae production.

Substitutability of coagulation process by pre-treatment of coagulation･flotation using natural algae coagulant

Substitutability of coagulation process by pre-treatment of coagulation･flotation using natural algae coagulant

Spatial competition mathematical model analysis for the invasion, removal of Kappaphycus Algae in gulf of Mannar with propagation delays

The aim of this paper is to study the impact of manual removal of Kappaphycus Alvarezii (KA) from the corals in the region of Gulf of Mannar (GoM) and the spatial competition between KA and Native Algae (NA) on coral with propagation delays of KA and NA. A non-linear system of ordinary differential equation (ODE) model has been developed to study the linear stability and the impact of delay terms in the stability at an equilibrium point.https://doi.org/10.28919/cmbn/3317

Ecological tipping points for an invasive kelp in rocky reef algal communities

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 587:93-104 (2018) - DOI: https://doi.org/10.3354/meps12429 Ecological tipping points for an invasive kelp in rocky reef algal communities David R. Schiel1,*, Stacie A. Lilley1, Paul M. South2 1Marine Ecology Research Group, School of Biological Sciences, University of Canterbury, Christchurch, New Zealand 2Cawthron Institute, Nelson, New Zealand *Corresponding author: david.schiel@canterbury.ac.nz ABSTRACT: Invasive species are affecting coastal ecosystems worldwide and there are many potential mechanisms that allow their spread into native communities. To investigate this phenomenon, we used an experiment in which the canopy of the southern bull kelp Durvillaea antarctica was removed in 2 seasons and community development was followed over 3 yr. The invasive kelp Undaria pinnatifida recruited almost exclusively into plots from which the natural canopy had been removed. We examined, through a series of regression tree models and change point analyses, the gradients of community responses that allowed the successful recruitment of this invasive species. Analyses revealed a range of coinciding conditions, especially a decline in fucoid cover below 20% and an increase in turf cover above 80%, that facilitated Undaria recruitment. The abundance of molluscan grazers and cover of subcanopy algae had lesser effects on Undaria recruitment. In an ecological sense, there was a clear tipping point in the interaction between canopy loss and the subsequent expansion of coralline turf that allowed communities to switch from dominance by native species to seasonal dominance of Undaria. This study illustrates the complex nature of disturbance thresholds and interactions within the native communities that can facilitate the spread and recruitment success of Undaria. KEY WORDS: Tipping point · Threshold · Rocky reef · Native algae · Invasive algae · Regression tree Full text in pdf format PreviousNextCite this article as: Schiel DR, Lilley SA, South PM (2018) Ecological tipping points for an invasive kelp in rocky reef algal communities. Mar Ecol Prog Ser 587:93-104. https://doi.org/10.3354/meps12429 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 587. Online publication date: January 25, 2018 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2018 Inter-Research.