Antioxidative Properties and Nutritional Contents of Selected Indigenous Microalgae

Microalgae are known to contain various beneficial pigments and are high in nutritional contents. The present studies were done to identify and explore the potential of indigenous microalgae as new natural sources for antioxidants with superior proximate and biochemical values. Fourteen microalgae were isolated, purified and cultured from fresh and brackish waters. The ability of microalgae as natural resources of antioxidants were studied through screening test using three antioxidative chemical assays (ferric thiocyanate (FTC), thiobarbituric acid (TBA) and 1,1’–diphenyl-2-picrylhydrazyl (DPPH)). Preliminary screening results showed that methanol extracts from six microalgae (Isochrysis galbana, Chaetoceros calcitrans, Scenedesmus quadricauda, Chlorella vulgaris, Nannochloropsis oculata and Tetraselmis tetrathele) were active in inhibiting the lipid peroxidation of linoleic acid. Among all the microalgae, I. galbana and C. calcitrans showed highest antioxidative activities (>90%) in FTC and TBA assays indicating possibility of active constituents for the protection from lipid peroxidation. Selected microalgae with high antioxidant values (I. galbana and C. calcitrans) were partitioned into different solvent fractions (hexane, dichloromethane, ethyl acetate, butanol and aqueous extracts) to separate components of wide mixture into group of compounds based on polarity and were re-tested with the antioxidative chemical assays. Dichloromethane extracts of I. galbana and C. calcitrans showed highest level of antioxidative activities with 97.1±0.1% and 97.4±0.1% linoleic acid peroxidation (LAP) inhibition in FTC assays respectively. Similar trends of high level antioxidative activities in these two microalgae were obtained in TBA assays. Effects of selected microalgal extracts on up-regulation of antioxidant enzymes gene expression in murine monocytic macrophage (RAW 264.7) cell lines were studied. Both dichloromethane extracts of I. galbana and C. calcitrans showed high up-regulation in the expression of all antioxidant enzymes genes tested in RAW 264.7. Liquid chromatography-mass spectrometry was employed to identify the constituents which were responsible for all of the antioxidative activities. Fucoxanthin and its isomers were identified as the major constituents in both microalgal species. Finally, nutritional analysis (proximate and biochemical) were done on microalgae with high antioxidative activities (I. galbana and C. calcitrans) in order to investigate their nutritive values. Both microalgae were found to be rich in nutrients with protein: carbohydrate: lipid percentage composition of 47.9±2.5: 26.8±0.2: 14.5±1.4 for I. galbana and 36.4±1.7: 27.4±3.0: 15.5±0.9 for C. calcitrans respectively. Both species contained high level of n-3 HUFA (28.0%±0.7: I. galbana and 28.5%±1.4: C. calcitrans) and n-6 HUFA (6.5%±1.8: I. galbana and 23.0%±2.5: C. calcitrans). Amino acid analyses also showed both microalgae contained high composition of essential amino acids. This study illustrated that some microalgae such as I. galbana and C. calcitrans have the potential for effective natural sources of antioxidants with high nutritional values

Disruption of bacterial cell-to-cell communication by marine organisms and its relevance to aquaculture.

Bacterial disease is one of the most critical problems in commercial aquaculture. Although various methods and treatments have been developed to curb the problem, yet they still have significant drawbacks. A novel and environmental-friendly approach in solving this problem is through the disruption of bacterial communication or quorum sensing (QS). In this communication scheme, bacteria regulate their own gene expression by producing, releasing, and sensing chemical signals from the environment. There seems to be a link between QS and diseases through the regulation of certain phenotypes and the induction of virulence factors responsible for pathogen-host association. Several findings have reported that numerous aquatic organisms such as micro-algae, macro-algae, invertebrates, or even other bacteria have the potential to disrupt QS. The mechanism of action varies from degradation of signals through enzymatic or chemical inactivation to antagonistic as well as agonistic activities. This review focuses on the existing marine organisms that are able to interfere with QS with potential application for aquaculture as bacterial control

Significance of microalgal–bacterial interactions for aquaculture.

Knowledge on the importance of associations between bacteria and microalgae in aquatic ecosystems is rather limited at the moment, mostly due to a lack of studies at the molecular and biochemical level of microorganisms. This paper discusses the current knowledge on microalgae–bacteria interactions and their potential impacts on the productivity, efficiency and sustainability of aquaculture. Current findings suggest that the interactions are complex and specific. The release of stimulatory products by bacteria that enhance the growth of microalgae, and vice versa, indicates the existence of mutualistic relations. Other factors, such as signalling between bacteria and microalgae, may also play an important role. Although these interactions may be of significant importance, to date, only a few findings have been reported on the use of consortia consisting of microalgae and bacteria for practical purposes. Interestingly, these results pointed out that a combination of microalgae and bacteria is often better than using either of them alone. Further research is needed to obtain a thorough understanding of the mechanisms behind the interactions between these microorganisms, including the identification of active compounds. This knowledge will enable the selection of appropriate consortia for different applications in aquaculture, including disease control and high and sustainable production of feed

Differences in feeding habits influence the growth performance and feeding efficiencies of African catfish (Clarias gariepinus) and lemon fin barb hybrid (Hypsibarbus wetmorei ♂ × Barboides gonionotus ♀) in a glycerol-based biofloc technology system versus a recirculating system

African catfish (Clarias gariepinus) and lemon fin barb hybrid (LFBH) (Hypsibarbus wetmorei ♂ × Barbodes gonionotus ♀) were cultured in either a biofloc technology (BFT) system or an individual recirculating aquaculture system (RAS) and their survival, growth, feeding efficiencies and biochemical composition were then compared after 8 weeks. LFBH and African catfish were chosen based on their different feeding habits. In the BFT treatments, glycerol was added to create a carbon to nitrogen ratio of 15, while the RAS system consisted of a fine mesh mechanical and biological filter. Each of the four treatments was triplicated, with each replicate consisting of 20 African catfish (0.98 ± 0.05 g) or LFBH (1.77 ± 0.02 g). Water quality and biofloc formation were examined weekly while the biofloc proximate composition was measured in weeks 5 and 8. During weeks 6, 7 and 8 the total viable bacterial colony forming units (CFU) were quantified. Regardless of species, biofloc formation was similar but the crude protein, lipid and ash significantly decreased from week 5 to week 8. However, biofloc crude protein and ash were significantly higher in the LFBH treatment. Total viable CFU were significantly higher (P < 0.05) in BFT compared to RAS, with LFBH having significantly more (P < 0.05) than African catfish. The proximate composition of the fish were unaffected by either system. Implementing BFT significantly (P < 0.05) improved both African catfish and LFBH growth, but this improvement was substantially higher for LFBH (41.6%) than for African catfish (7.6%). This may be due to a better ability of LFBH to consume smaller particles. Preliminary results also indicate that the nutritional composition of bioflocs could be influenced by biofloc age, which could have implications to solids management

Growth and survival of bilateral eyestalk ablated small male freshwater prawn Macrobrachium rosenbergii (DeMan)

The freshwater prawn Macrobrachium rosenbergii exhibits three male morphotypes: Blue Claw (BC) male, Orange Claw (OC) male and Small Male (SM). Effects of bilateral eyestalk ablation on growth and survival of SM freshwater prawn were determined in monosex culture. The SM juveniles (9 g) were used in treatment bilateral eyestalk ablation and control (intact/unablated) at a hatchery. Ablated prawns were stocked at 5 prawn m-2 for 80 days. At the end of the experimental period, the average weight increased from 8.96±1.06 to 40.95±4.01 g, with bilaterally ablated prawns attaining the greatest growth. The final body weight of prawn at harvest was 40.95± 4.01 g in bilateral ablated SM and 22.69±2.41 g in unablated SM. The specific growth rate of bilaterally ablated prawns (1.90%) was significantly higher than unablated prawns (1.15%). The growth trend of SM prawn was increased significantly from the controls. The wet weight of ablated prawns doubled compared to the control but yield was not significantly different due to mortality. The survival of bilaterally ablated prawn was significantly low 50%. The potency of bilateral eyestalk ablation in enhancing growth rates may be limited due to high mortalities of ablated prawn

PROBIOTICS BACTERIA AS QUORUM SENSING DEGRADER CONTROL Aeromonas hydrophila PATHOGENICITY IN CULTURED RED HYBRID TILAPIA

Quorum sensing (QS) is the interaction of bacteria cell-to-cell to regulate various bacterial functions, including bacterial virulence. It has been suggested that QS interruption is an anti-infective strategy to combat diseases in aquaculture. This research was conducted to isolate the potential probiotics bacteria as QS degrader from the fish gut and investigate its efficiency in reducing Aeromonas hydrophila pathogenicity by in vitro and in vivo assay. In this study, three isolates from the fish gut were able to degrade N-acyl homoserine lactone (AHL), one of the QS signals of A. hydrophila. Based on 16S rDNA sequence analysis, isolate CPi12 was identified as Klebsiella sp., CBa5 and CBa7 were identified as Enterobacter tabaci. These isolates were co-cultured with A.hydrophila for five days. Results revealed that all isolates could decrease the AHL production of A. hydrophila but did not affect the growth of the pathogen. In vivo assay results showed that isolate CPi12 reduced the pathogenicity of A.hydrophila against tilapia with a significantly higher survival rate (p&lt;0.05) of fish in the CPi12-fed group than the control group. Meanwhile, isolate CBa7 was significant (p&lt;0.05) for growth performance, including specific growth rate (SGR), weight gain (WG), feed conversion ratio (FCR), mean weight gain (MWG), and average daily growth (ADG) among whole treatment and control group. These results displayed that the probiotics bacteria as QS degrader isolated from the fish gut could control the pathogenicity of Aeromonas hydrophila. It has been proposed that QS degrader bacteria might be an alternative solution for disease control

Effects of stocking density on growth performance and survival of three male morphotypes in all-male culture of Macrobrachium rosenbergii (De Man)

A study was conducted to appraise the effect of different stocking densities on three male morphotypes named blue claw (BC), orange claw (OC) and small male (SM) at harvest in all-male culture and to assess the growth performance of BC, OC and SM in each isolated culture. Trials involving three stocking densities of all-male prawn viz., 20, 30 and 40 juvenile m-2 were carried out in replicates. After 4 months of culture, BC, OC and SM were sorted from all tanks and restocked at 5 m-2 in treatments BC, OC and SM respectively for 80 days. A difference in prawn density significantly (p<0.05) affected adversely on morphotypes. The highest survival rate combined with good yield performance was from 20 juvenile m-2 stocking density with 21% BC, 62.5% OC and 16.5% SM, respectively. In isolation culture, the average specific growth rate of the SM population (1.22) was significantly higher than that of the OC (1.01) and the BC (0.43) population. The survival of the SM population was 100% while for others it was 72%. Absolute weight of prawn was significantly greater in the male OC (23.87 g) than the SM (19.57 g) and the BC males (6.31 g). Impacts of isolated culture on population structure were much more pronounced in the SM population than others

Effects of micro-algae commonly used in aquaculture on acyl-homoserine lactone quorum sensing.

Quorum sensing, bacterial cell-to-cell communication with small signal molecules such as acyl-homoserine lactones, regulates the virulence of many pathogenic bacteria. Therefore, interfering with quorum sensing is currently being explored as a novel biocontrol strategy to fight bacterial infections. In this study, the effects of 19 micro-algal strains on acyl-homoserine lactone-regulated phenotypes of three reporter strains were investigated. Two freshwater micro-algae inhibited violacein production of quorum sensing reporter strain Chromobacterium violaceum CV026. Further tests using Escherichia coli JB523 showed that micro-algal extracts inhibited or stimulated quorum sensing, depending on the algal strain. One freshwater and five marine algae showed quorum sensing inhibitory activity, whereas two algae stimulated quorum sensing-regulated gene expression. Micro-algal strains that showed inhibitory activity in the previous assays also inhibited acyl-homoserine lactone-regulated bioluminescence in the aquaculture pathogen Vibrio harveyi. The growth of all reporter strains was found to be unaffected by the micro-algal samples. The most promising micro-algal strain was found to be Chlorella saccharophila CCAP211/48, as its extracts inhibited quorum sensing-regulated gene expression in all three reporter strains

Screening of Malaysian indigenous microalgae for antioxidant properties and nutritional value

Fourteen indigenous microalgal samples from Malaysia were isolated, purified and cultured from fresh,brackish and marine waters. The ability of the microalgae to be natural sources of antioxidants was studied by a screening test using three antioxidant chemical assays[ferric thiocyanate (FTC), thiobarbituric acid (TBA) and 1, 1’-diphenyl-2-picrylhydrazyl (DPPH)]. The results showed that six microalgal methanolic crude extracts(Isochrysis galbana, Chaetoceros calcitrans, Scenedesmus quadricauda, Chlorella vulgaris, Nannochloropsis oculata and Tetraselmis tetrathele) were active in inhibiting the lipid peroxidation of linoleic acid. Among all the microalgae, I. galbana and C. calcitrans showed the highest antioxidant activity (>90%) in FTC and TBA assays, indicating that these microalgae might contain active compounds for protection from lipid peroxidation. Nutritional analyses were performed on microalgae with high antioxidant activities (I. galbana and C. calcitrans) in order to investigate their nutritive value. Both microalgae were found to be rich in nutrients. For examples, I. galbana had average percentage composition of protein, carbohydrate,and lipid, as 47.9±2.5; 26.8±0.2; 14.5±1.4%, respectively,while the corresponding values for C. calcitrans were 36.4± 1.7; 27.4±3.0; 15.5±0.9%. In addition, they contained high levels of omega-3 polyunsatrated fatty acids (PUFA)(28.0%±0.7 in I. galbana and 28.5%±1.4 in C. calcitrans),omega-6 PUFA (6.5%±1.8 in I. galbana and 23.0%±2.5 in C. calcitrans) and a high composition of essential amino acids. This study illustrates that some microalgae such as I. galbana and C. calcitrans have the potential to be used as natural sources of antioxidants with high nutritional value

Effect of cold shock and hormone on growth and male morphotypes of freshwater prawn, Macrobrachium rosenbergii (de Man)

A study was carried out to determine the effects of cold shock and 17α-methyl testosterone hormone on growth in male morphotypes of giant freshwater prawn Macrobrachium rosenbergii. Juvenile males were exposed at 18 ± 0.25°C for 24 h with normal feed and cultured at 27 ± 0.7°C for 70 days as well as administrated feed with hormone separately at 400 mg kg−1 feed for the first 30 days of culture period (70 days). The growth (Mean ± SD) of male juveniles at harvest was closely homogenous in the cold shock (CLS) treated group; while it was heterogeneous in hormone-treated (MH) and control (CO) groups. Although total body weight of blue claw (BC) males was not significantly different in the controls and the treatments, the 2nd pereiopods (claw) weight and length of the BC was significantly lower in CLS (0.52 ± 0.05 g, 10.02 ± 0.65 cm) than that in the CO (0.71 ± 0.16 g, 12.53 ± 0.57 cm) and MH (0.75 ± 0.08 g, 12.85 ± 1.18 cm) treatments. Survival of prawns was similar in treatments. The morphotypes of male prawn were significantly different with 59% of BC males with small-sized claws in CLS and, 45% and 30% of BC males with large-sized claws in MH and CO respectively. Cold shock adversely affected male morphotypes of the prawn so that numbers of BC males with small-sized claw was the highest in CLS tank. However, the CLS treatment resulted in more uniform marketable prawns and suggested a high production of BC males in an all-male culture system