Ravi S. Baghel

Selenium and spermine alleviate cadmium induced toxicity in the red seaweed Gracilaria dura by regulating antioxidants and DNA methylation.

The protective role of exogenously supplied selenium (Se) and polyamines (PAs) such as putrescine (Put) and spermine (Spm) in detoxifying the cadmium (Cd) induced toxicity was studied in the marine red alga Gracilaria dura in laboratory conditions. The Cd exposure (0.4 mM) impede the growth of alga while triggering the reactive oxygen species (ROS viz. O(2)(•-) and H(2)O(2)) generation, inhibition of antioxidant system, and enhancing the lipoxygenase (LOX) activity, malondialdehyde (MDA) level and demethylation of DNA. Additions of Se (50 μM) and/or Spm (1 mM) to the culture medium in contrast to Put, efficiently ameliorated the Cd toxicity by decreasing the accumulation of ROS and MDA contents, while restoring or enhancing the level of enzymatic and nonenzymatic antioxidants and their redox ratio, phycobiliproteins and phytochelatins, over the controls. The isoforms of antioxidant enzymes namely superoxide dismutase (Mn-SOD, ~150 kDa; Fe-SOD ~120 kDa), glutathione peroxidase (GSH-Px, ~120 and 140 kDa), glutathione reductase (GR, ~110 kDa) regulated differentially to Se and/or Spm supplementation. Furthermore, it has also resulted in enhanced levels of endogenous PAs (specially free and bound insoluble Put and Spm) and n-6 PUFAs (C20-3, n-6 and C20-4, n-6). This is for the first time wherein Se and Spm were found to regulate the stabilization of DNA methylation by reducing the events of cytosine demethylation in a mechanism to alleviate the Cd stress in marine alga. The present findings reveal that both Se and Spm play a crucial role in controlling the Cd induced oxidative stress in G. dura.

Biorefining of marine macroalgal biomass for production of biofuel and commodity chemicals.

The large scale production of marine macroalgae, mainly for human consumption, has given rise to their consideration as a non-lignocellulosic feedstock for the production of renewable fuels. However, making biofuel economical from algal biomass requires the co-production of additional useful biochemical components that are unique to algae and that have a proven market value. A viable and sustainable biorefining technology that maximizes the utilisation of feedstock for the production of chemicals along with fuel is, therefore, indispensable. Here, we for the first time demonstrate a tractable integrated process that facilitates sequential extraction of the major components of red algal biomass as commodity products such as pigments, lipid, agar, minerals and energy dense substrate (cellulose). The computed yield data from small-scale biorefinery trials suggest that a ton of fresh biomass supplies several valuable extracts: 0.3–0.7 kg of R-phycoerythrin (R-PE), 0.1–0.3 kg of R-phycocyanin (R-PC), 1.2–4.8 kg of lipids, 28.4–94.4 kg of agar, 4.4–41.9 kg of cellulose and 3.1–3.6 kiloliters of mineral solution. The enzymatic hydrolysis and fermentation of cellulose thus obtained would yield 1.8–17.4 kg of ethanol. A distinct advantage of this process over direct extraction is the improved quality of agar (gel strength higher by 1.5–3 fold) without alkali and acid pretreatment of sample, the elimination of residue and the reduction by up to 85% in chemicals usage in cellulose extraction. The findings reported in this study forms the basis for starting new ocean-based bio-industries minimizing the dependence on the terrestrial resources for food, feed, energy and chemicals.

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.

Role of bacterial isolates in enhancing the bud induction in the industrially important red alga Gracilaria dura.

Plant growth depends on the integration of environmental cues, nitrogen fixation and phytohormone-signaling pathways. The growth and development of Gracilaria dura was significantly influenced by the association of bacterial isolates. The putative bud-inducing epiphytic Exiguobacterium homiense and endophytic Bacillus pumilus, Bacillus licheniformis were examined for their ability to fix nitrogen and produce indole-3-acetic acid (IAA). These bacterial isolates were identified to the species level by biochemical tests, fatty acid and partial 16S rRNA gene sequence analysis. The B. pumilus, B. licheniformis and E. homiense produced 445.5, 335 and 184.1 μg mL(-1) IAA and 12.51, 10.14 and 6.9 mM mL(-1) ammonium, respectively, as determined using HPLC and spectroscopy. New bud regeneration observed after the addition of total protein of the bacterial isolates suggests that IAA is conjugated with protein. The epi- and endophytic bacterial isolates were able to induce five and 10 new buds per frond, respectively, in comparison to the control, where one to two buds were observed. The combination of 25 °C and 30‰ showed the optimum condition for bud induction in G. dura when incubated with the total protein of B. pumilus. Our finding revealed for the first time that IAA coupled with nitrogen fixation induce and regenerate new buds in G. dura.

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.

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.

Chapter Two - Seaweed Metabolomics: A New Facet of Functional Genomics

Abstract Metabolomics is one of the emerging areas of functional genomics and provides newer insights in systems biology. The integration of metabolome with transcriptome analysis facilitates our understanding of gene functionality and its regulation in various metabolic pathways. Marine organisms have a variety of unique biological processes and adaptations suitable for their successful propagation. Marine macroalgae, known as ‘seaweeds’, are one of the economically important renewable resources of the oceans and have characteristic morphological and physiological processes quite different from terrestrial plants. Seaweeds being attached forms in intertidal region undergo periodic diverse chronic stresses arising from variations in desiccation, irradiance, ultraviolet radiation, salinity, tidal currents and others from anthropogenic activities. Despite the advancement in transcriptomics for seaweeds in recent times, the genetic regulations controlling various biochemical pathways are still in its inception and largely remain unexplored. The study of metabolomics provides a snapshot of cell’s catalytic and regulatory processes and also deciphers metabolic responses involved in plant and environment interactions. While summarizing the recent advancements made in analytical technology platforms, a comprehensive review of metabolomics was prepared and discussed from the context of functional genomics, systems biology and biotechnology to gain newer insights into various regulatory networks functioning in the seaweeds.

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.

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.