Seaweed-based cellulose: Applications, and future perspectives.

Cellulose is a naturally occurring organic polymer extracted mainly from lignocellulosic biomass of terrestrial origin. However, the increasing production of seaweeds for growing global market demands has developed the opportunity to use it as an additional cellulose source. This review aims to prepare comprehensive information to understand seaweed cellulose and its possible applications better. This is the first review that summarizes and discusses the cellulose from all three types (green, red, and brown) of seaweeds in various aspects such as contents, extraction strategies, and cellulose-based products. The seaweed cellulose applications and future perspectives are also discussed. Several seaweed species were found to have significant cellulose content (9-34% dry weight). The review highlights that the properties of seaweed cellulose-based products were comparable to products prepared from plant-based cellulose. Overall, this work demonstrates that cellulose could be economically extracted from phycocolloids industrial waste and selected cellulose-rich seaweed species for various commercial applications.

Synthesis, characterization and application of green seaweed mediated silver nanoparticles (AgNPs) as antibacterial agents for water disinfection.

A simple and eco-friendly method for the synthesis of hybrid bead silver nanoparticles (AgNPs) employing the aqueous extract derived from natural and renewable source namely tropical benthic green seaweed Ulva flexuosa was developed. This route involves the reduction of Ag+ ions anchored onto macro porous methacrylic acid copolymer beads to AgNPs for employing them as antibacterial agents for in vitro water disinfection. The seaweed extract itself acts as a reducing and stabilizing agent and requires no additional surfactant or capping agent for forming the AgNPs. The nanoparticles were analyzed using high-resolution transmission electron microscopy, UV-Vis spectroscopy, Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive X-ray analysis and inductively coupled plasma optical emission spectroscopy. The study elucidates that such biologically synthesized AgNPs exhibit potential antibacterial activity against two Gram positive (Bacillus subtilis, Staphylococcus aureus) and two Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacterial strains tested. The bacterial count in treated water was reduced to zero for all the strains. Atomic force microscopy was performed to confirm the pre- and post-state of the bacteria with reference to their treatment with AgNPs. Attributes like facile environment-friendly procedure, stability and high antibacterial potency propel the consideration of these AgNPs as promising antibacterial entities.

Integration of protein extraction with a stream of byproducts from marine macroalgae: A model forms the basis for marine bioeconomy.

The present study describes an advanced biorefinery model for marine macroalgae that assumes significant importance in the context of marine bio-economy. The method investigated in this study integrates the extraction of crude proteins with recovery of minerals rich sap, lipids, ulvan and cellulose from fresh biomass of Ulva lactuca. The protein content extracted was 11±2.12% on dry weight basis with recovery efficiency of 68.75±4.01%. The amino acid composition of crude protein fraction showed iso-leucine as the most abundant amino acid with 16.51±0.03% followed by histidine, arginine, tyrosine, serine, aspartic acid, threonine, phenyl alanine, leucine, alanine, lysine, glycine and glutamic acid (0.22±0.24%). The digestibility of protein was as high as 85.86±5.92% indicating its suitability for use in food supplements. The protein production with co-recovery of other products would not only result in effective utilisation marine macroalgal resources but also forms the basis for marine bio-economy.

Synthesis and characterization of seaweed cellulose derived carboxymethyl cellulose.

In the present study, cellulose (SWC) extracted from green seaweed Ulva fasciata was processed to synthesize carboxymethyl cellulose (SWCMC). The seaweed cellulose (∼15% DW) was first processed for α cellulose extraction (10.1% on DW) followed by the synthesis and characterization of SWCMC. Thin films were prepared using commercial CMC (CCMC), SWCMC and SWCMC-metal nanoparticle (2% wt/v) by solvent evaporation technique. Films were studied for molecular weight, degree of carboxylation, viscosity and characterized by FT-IR and TGA. AFM surface morphology of SWCMC-metal nanoparticle film confirms the uniform distribution of sphere shaped metal nanoparticle on the film surface with the size in the range of 50-75nm. Further, SWCMC film showed antimicrobial activity when prepared with Ag and leaf extract of Azadirachta indica. The biodegradable nature of SWCMC film was confirmed by growing marine fungus Cladosporium spherospermum on CMC agar plates. Thus, SWCMC films exhibit potential applications in cosmetic, food, textiles, medical, agricultural and pharmaceutical industries.

Marine Microbes as a Potential Source of Cellulolytic Enzymes.

Marine environment hosts the wide range of habitats with remarkably high and diverse microbial populations. The ability of marine microorganisms to survive in extreme temperature, salinity, and pressure depends on the function of multivarious enzyme systems that in turn provide vast potential for biotechnological exploration studies. Therefore, the enzymes from marine microorganism represent novel bio catalytic potential with stable and reliable properties. Microbial cellulases constitute a major group of industrial enzymes that find applications in various industries. Majority of cellulases are of terrestrial origin, and very limited research has been carried out to explore marine microbes as a source of cellulases. This chapter presents an overview about the types of marine polysaccharases, classification and potential applications of cellulases, different sources of marine cellulases, and their future perspectives.

An integrated process for the extraction of fuel and chemicals from marine macroalgal biomass.

We describe an integrated process that can be applied to biomass of the green seaweed, Ulva fasciata, to allow the sequential recovery of four economically important fractions; mineral rich liquid extract (MRLE), lipid, ulvan, and cellulose. The main benefits of our process are: a) its simplicity and b) the consistent yields obtained from the residual biomass after each successive extraction step. For example, dry Ulva biomass yields ~26% of its starting mass as MRLE, ~3% as lipid, ~25% as ulvan, and ~11% as cellulose, with the enzymatic hydrolysis and fermentation of the final cellulose fraction under optimized conditions producing ethanol at a competitive 0.45 g/g reducing sugar. These yields are comparable to those obtained by direct processing of the individual components from primary biomass. We propose that this integration of ethanol production and chemical feedstock recovery from macroalgal biomass could substantially enhance the sustainability of marine biomass use.

A simple process for recovery of a stream of products from marine macroalgal biomass.

The present study describes a simple process for recovering a stream of products sequentially including bioethanol from the fresh biomass of the red seaweed Gracilaria corticata. From processing of 100g fresh biomass (∼12.2 g dry), 166 ± 3 µg/g R-phycoerythrin, 126±4µg/g R-phycocyanin can be realized on fresh weight basis, and 1.41 ± 0.03% crude lipid, 22.45 ± 0.53% agar, 12.39 ± 0.85% soil conditioner, 2.89 ± 0.04% bioethanol on dry weight basis along with 318 ± 3 ml of mineral rich liquid with possible fertilizer applications. The advantages of this process are complete utilization of feedstock without compromising the yield and quality of products, reusability of solvents and no solid waste. Further, the products recovered from one ton fresh biomass were found to have an estimated market value of USD 1051 while processing cost including raw material as 241 USD, a fourfold value addition of feedstock.

Methyl Jasmonate-Induced Lipidomic and Biochemical Alterations in the Intertidal Macroalga Gracilaria dura (Gracilariaceae, Rhodophyta).

The role of exogenously added methyl jasmonate (MeJA), a lipid-derived signaling compound, in inducing oxidative stress in the marine red macroalga Gracilaria dura was investigated. MeJA at a concentration of 1-100 µM was a strong stimulant of reactive oxygen species (H(2)O(2), HO· and O(2) (·-)) (P < 0.05) causing considerable oxidative stress in G. dura. This further led to lipid peroxidation and degradation of the pigments Chl a and phycocyanin, with a concomitant increase in phycoerythrin. The MeJA-induced oxidative burst also led to the induction of a fatty acid oxidation cascade, resulting in the synthesis of hydroxy-oxylipins and the up-regulation of the 13-lipoxygenase pathway. Electrospray ionization-mass spectrometry-based shotgun lipidomic analysis revealed that monogalactosyldiacylglycerol (a chloroplastic glycerolipid) and phosphatidylcholine (extrachloroplastidic phopholipid) were the most affected lipid classes. The degradation of 18:3-fatty acid-containing monogalactosyldiacylglycerol inferred that it provided fatty acyl chains for the biosynthesis of 13-hydroperoxylinolenic acid, which was further directed towards either the jasmonate pathway or other alternative pathways of the fatty acid oxidation cascade, analogous to higher plants. Also, G. dura modulated the lipid acyl chains in such a way that no significant change was observed in the fatty acid profile of the treated thalli as compared with those of the control, except for C16:0, C16:1 (n-9), C20:3 (n-6) and C20:4 (n-6) (P < 0.05). Furthermore, MeJA caused the accumulation of phenolic compounds and the up-regulation of enzymes involved in secondary metabolism such as polyphenol oxidase, shikimate dehydrogenase and phenylalanine ammonia-lyase, indicating a shift towards secondary metabolism as a defense strategy to combat the induced oxidative stress.

Production of genetically and developmentally modified seaweeds: exploiting the potential of artificial selection techniques.

Plant feedstock with specific, modified developmental features has been a quest for centuries. Since the development and spread of agriculture, there has been a desire for plants producing disproportionate-or more abundant and more nutritional-biomass that meet human needs better than their native counterparts. Seaweed aquaculture, targeted for human consumption and the production of various raw materials, is a rapidly expanding field and its stakeholders have increasing vested interest for cost-effective and lucrative seaweed cultivation processes. Thus, scientific research on seaweed development is particularly timely: the potential for expansion of seaweed cultivation depends on the sector's capacity to produce seaweeds with modified morphological features (e.g., thicker blades), higher growth rates or delayed (or even no) fertility. Here, we review the various technical approaches used to modify development in macroalgae, which have attracted little attention from developmental biologists to date. Because seaweed (or marine macroalgae) anatomy is much less complex than that of land plants and because seaweeds belong to three different eukaryotic phyla, the mechanisms controlling their morphogenesis are key to understanding their development. Here, we present efficient sources of developmentally and genetically modified seaweeds-somatic variants, artificial hybrids and mutants-as well as the future potential of these techniques.

Effect of quorum sensing signals produced by seaweed-associated bacteria on carpospore liberation from Gracilaria dura.

Epiphytic and endophytic bacteria associated with green macroalgae Ulva (U. fasciata and U. lactuca) and red macroalgae Gracilaria (G. corticata and G. dura) have been identified from three different seasons to evaluate the effect of quorum sensing (QS) molecules on carpospores liberation from Gracilaria dura. The bacterial isolates belonging to the orders Bacillales, Pseudomonadales, Alteromonadales, and Vibrionales were present in all seasons, whereas Actinomycetales and Enterobacteriales were confined to pre-monsoon and post-monsoon seasons, respectively. Among all the Gram-negative bacteria, seven isolates were found to produce different types of N-acyl homoserine lactones (AHLs). Interestingly, Shewanella algae produced five types of AHL: C4-HSL, HC4-HSL, C6-HSL, 3-oxo-C6-HSL, and 3-oxo-C12-HSL. Subsequently, the AHLs producing bacterial isolates were screened for carpospore liberation from G. dura and these isolates were found to positively induce carpospore liberation over the control. Also, observed that carpospore liberation increased significantly in C4- and C6-HSL treated cystocarps. Sodium dodecyl sulfate and native polyacrylamide gel electrophoresis of the total protein of the C4- and C6-HSL treated cystocarps showed two specific peptide bands of different molecular weights (50 kDa and 60 kDa) as compared to the control, confirming their indirect effect on carpospore liberation.

Antimicrobial compounds from seaweeds-associated bacteria and fungi.

In recent decade, seaweeds-associated microbial communities have been significantly evaluated for functional and chemical analyses. Such analyses let to conclude that seaweeds-associated microbial communities are highly diverse and rich sources of bioactive compounds of exceptional molecular structure. Extracting bioactive compounds from seaweed-associated microbial communities have been recently increased due to their broad-spectrum antimicrobial activities including antibacterial, antifungal, antiviral, anti-settlement, antiprotozoan, antiparasitic, and antitumor. These allelochemicals not only provide protection to host from other surrounding pelagic microorganisms, but also ensure their association with the host. Antimicrobial compounds from marine sources are promising and priority targets of biotechnological and pharmaceutical applications. This review describes the bioactive metabolites reported from seaweed-associated bacterial and fungal communities and illustrates their bioactivities. Biotechnological application of metagenomic approach for identifying novel bioactive metabolites is also dealt, in view of their future development as a strong tool to discover novel drug targets from seaweed-associated microbial communities.

Unraveling the Functions of the Macroalgal Microbiome.

Macroalgae are a diverse group of photosynthetic eukaryotic lower organisms and offer indispensable ecosystem services toward sustainable productivity of rocky coastal areas. The earlier studies have mainly focused on elucidation of the roles of the epiphytic bacterial communities in the ecophysiology of the host macroalga. However, mutualistic interactions have become topic of current interest. It is evident from recent studies that a fraction of epiphytic bacterial communities can be categorized as "core microbial species", suggesting an obligate association. Epiphytic bacterial communities have also been reported to protect macroalgal surfaces from biofouling microorganisms through production of biologically active metabolites. Because of their intrinsic roles in the host life cycle, the host in turn may provide necessary organic nutrients in order to woo pelagic microbial communities to settle on the host surfaces. However, the precise composition of microbiomes and their functional partnership with hosts are hardly understood. In contrast, the microbial studies associated with human skin and gut and plants have significantly advanced our knowledge on microbiome and their functional interactions with the host. This has led to manipulation of the microbial flora of the human gut and of agricultural plants for improving health and performance. Therefore, it is highly imperative to investigate the functional microbiome that is closely involved in the life cycles of the host macroalgae using high-throughput techniques (metagenomics and metatranscriptomics). The findings from such investigations would help in promoting health and productivity in macroalgal species through regulation of functionally active microbiome.

Characterization of agarophytic seaweeds from the biorefinery context.

The major seaweed components such as natural colorants (R-phycoerythrin (R-PE), R-phycocyanin (R-PC)), minerals, proteins, lipids, cellulose and agar which are of considerable commercial value were estimated in 15 different agarophytic seaweeds. R-PE and R-PC contents ranged from 138.33 ± 17.67 to 1039.43 ± 27.65 µg/g and 50.26 ± 6.63 to 818.2 ± 48 µg/g on fresh weight (fw) basis, respectively. Appreciable amounts of both macro-minerals (K, Na, Ca, Mg) and micro-minerals (Fe, Zn, Se, Mn) were estimated. The total lipid and protein contents were 0.65 ± 0.06% to 1.53 ± 0.07% and 4.75 ± 0.5% to 19.31 ± 3.5% on dry weight (dw) basis respectively while cellulose and agar contents varied from 3.7 ± 0.13% to 12.20 ± 0.45% and 9.17 ± 0.62% to 25.23 ± 0.50% dw, respectively. The overall finding of this study enable the selection and value addition of agarophytic feedstock for biorefinery.

Seaweed-microbial interactions: key functions of seaweed-associated bacteria.

Seaweed-associated bacteria play a crucial role in morphogenesis and growth of seaweeds (macroalgae) in direct and/or indirect ways. Bacterial communities belonging to the phyla Proteobacteria and Firmicutes are generally the most abundant on seaweed surfaces. Associated bacterial communities produce plant growth-promoting substances, quorum sensing signalling molecules, bioactive compounds and other effective molecules that are responsible for normal morphology, development and growth of seaweeds. Also, bioactive molecules of associated bacteria determine the presence of other bacterial strains on seaweeds and protect the host from harmful entities present in the pelagic realm. The ecological functions of cross-domain signalling between seaweeds and bacteria have been reported as liberation of carpospores in the red seaweeds and settlement of zoospores in the green seaweeds. In the present review, the role of extracellular polymeric substances in growth and settlement of seaweeds spores is also highlighted. To elucidate the functional roles of associated bacteria and the molecular mechanisms underlying reported ecological phenomena in seaweeds requires a combined ecological, microbiological and biochemical approach.

Nitrate and phosphate regimes induced lipidomic and biochemical changes in the intertidal macroalga Ulva lactuca (Ulvophyceae, Chlorophyta).

This study was carried out in order to understand the lipid and biochemical alterations resulting from different nutritional regimes of nitrate and phosphate in Ulva lactuca. The algal thalli cultured in artificial seawater (ASW) showed higher levels of carbohydrates and non-polar lipids and increased phosphatase activities, accompanied by degradation of polar lipids, proteins and pigments. Further, higher levels of lipid hydroperoxides indicated reative oxygen species (ROS)-mediated non-enzymatic lipid peroxidation due to nutritional limitation-induced oxidative stress. Those thalli cultured in ASW supplemented with nitrate showed responses corresponding to nitrate addition, such as an increase in pigments, monogalactosyldiacylglycerols, polyunsaturated fatty acids and nitrate reductase. In addition, these thalli showed partial induction of phosphatases, low phospholipids, and high sulfolipid and 1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine (DGTS) due to phosphate limitation. Similarly, algal thalli cultured in ASW supplemented with phosphate showed down-regulation of phosphatases, an increase in phospholipids due to availability of phosphate as well as a decrease in nitrate reductase, pigment, monogalactosyldiacylglycerols and polyunsaturated fatty acids due to nitrate limitation. On the other hand, algal thalli cultured in ASW supplemented with both nitrate and phosphate showed recovery of lost pigments and proteins, a high monogalactosyldiacylglycerol/digalactosyldiacylglycerol ratio, high unsaturation and high oxylipin levels (both C18 and C20). Further, the accumulation of indole-3-acetic acid in nutrient-limited thalli and of kinetin and kinetin riboside in nutrient-supplemented thalli indicated their antagonistic roles under nutrient stress. Thus, U. lactuca copes with nitrate and phosphate nutritional stress by altering the metabolic pathways involved in lipid biosynthesis including a shift in lipid classes, fatty acids, oxylipins and indole-3-acetic acid/kinetin cross-talk.

Molecular phylogeny and barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA genes.

The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.

Enzymatic hydrolysis and production of bioethanol from common macrophytic green alga Ulva fasciata Delile.

The green seaweed Ulva which proliferates fast and occurs abundantly worldwide was used as a feedstock for production of ethanol following enzymatic hydrolysis. Among the different cellulases investigated for efficient saccharification, cellulase 22119 showed the highest conversion efficiency of biomass into reducing sugars than Viscozyme L, Cellulase 22086 and 22128. Pre-heat treatment of biomass in aqueous medium at 120°C for 1h followed by incubation in 2% (v/v) enzyme for 36 h at 45°C gave a maximum yield of sugar 206.82±14.96 mg/g. The fermentation of hydrolysate gave ethanol yield of 0.45 g/g reducing sugar accounting for 88.2% conversion efficiency. These values are substantially higher than those of reported so far for both agarophytes and carrageenophytes. It was also confirmed that enzyme can be used twice without compromising on the saccharification efficiency. The findings of this study reveal that Ulva can be a potential feedstock for bioethanol production.

Detection of ionic liquid stable cellulase produced by the marine bacterium Pseudoalteromonas sp. isolated from brown alga Sargassum polycystum C. Agardh.

An extracellular cellulase produced by marine bacterium Pseudoalteromonas sp. was studied for its activity and stability in six different ionic liquids (ILs) over a wide range of concentrations (1-20% v/v) and compared with aqueous medium as control. Enzyme showed its optimal activity at 45°C and at pH 5 in control. Although the activity varied with the type of IL and its concentration used, the activity measured at 5% (v/v) was maximum with [EMIM]Br followed by [EMIM]Ac, [BMIM]Cl, [C2MIM][CH3SO3], [BMIM][OTF] and [BMPL][OTF] with 115%, 104.7%, 102.2%, 98.33%, 93.84% and 92.67%, respectively, and >80% activity at 15% (v/v) in all ILs. The enzyme stability at 5% (v/v) IL concentration for 36h was superior to commercial cellulase. The cellulase activity enhanced by 1.35- to 1.72-fold over control when 5% (v/v) IL based reaction medium with algal biomass was used and thus showed potentials for saccharification of biomass in a single step process.

Bacterial extracellular polymeric substances and their effect on settlement of zoospore of Ulva fasciata.

The extracellular polymeric substances (EPSs) secreted by Bacillus flexus (GU592213) were estimated to have the molecular weight of approximately 1528 and 33,686 kDa with the elemental composition of Na, P, Mg, C, O, Cl and S. The (1)H NMR and FT-IR analysis of EPS confirmed the presence of different aliphatic and aromatic groups. The EPS was amorphous in nature with an average particle size of 13.969 µm (d 0.5) and roughness of 193 nm. The GC-MS analysis has revealed different monosaccharides such as fucose, ribose, xylose, galactose, mannose and glucose. Oligo and polysaccharides were detected with MALDI TOF-TOF MS. The bacterial EPS for the first time tested as a natural substratum for settle of zoospores of Ulva fasciata by incubating for various durations ranging from 2h to 48 h. The zoospore settlement on EPS coated cover slips progressively increased with incubation time in axenic cultures over controls. The EPS, thus investigated in this study was found to facilitate the primary settlement of spores that play crucial role in recruitment of macroalgal communities in coastal environment including intertidal regions.