Utilization of Clarified Butter Sediment Waste as a Feedstock for Cost-Effective Production of Biodiesel.

The rising demand and cost of fossil fuels (diesel and gasoline), together with the need for sustainable, alternative, and renewable energy sources have increased the interest for biomass-based fuels such as biodiesel. Among renewable sources of biofuels, biodiesel is particularly attractive as it can be used in conventional diesel engines without any modification. Oleaginous yeasts are excellent oil producers that can grow easily on various types of hydrophilic and hydrophobic waste streams that are used as feedstock for single cell oils and subsequently biodiesel production. In this study, cultivation of Rhodosporidium kratochvilovae on a hydrophobic waste (clarified butter sediment waste medium (CBM)) resulted in considerably high lipid accumulation (70.74% w/w). Maximum cell dry weight and total lipid production were 15.52 g/L and 10.98 g/L, respectively, following cultivation in CBM for 144 h. Neutral lipids were found to accumulate in the lipid bodies of cells, as visualized by BODIPY staining and fluorescence microscopy. Cells grown in CBM showed large and dispersed lipid droplets in the intracellular compartment. The fatty acid profile of biodiesel obtained after transesterification was analyzed by gas chromatography-mass spectrometry (GC-MS), while its quality was determined to comply with ASTM 6751 and EN 14214 international standards. Hence, clarified sediment waste can be exploited as a cost-effective renewable feedstock for biodiesel production.

Co-culturing of oleaginous microalgae and yeast: paradigm shift towards enhanced lipid productivity.

Oleaginous microalgae and yeast are the two major propitious factories which are sustainable sources for biodiesel production, as they can accumulate high quantities of lipids inside their bodies. To date, various microalgal and yeast species have been exploited singly for biodiesel production. However, despite the ongoing efforts, their low lipid productivity and the high cost of cultivation are still the major bottlenecks hindering their large-scale deployment. Co-culturing of microalgae and yeast has the potential to increase the overall lipid productivity by minimizing its production cost as both these organisms can utilize each other's by-products. Microalgae act as an O2 generator for yeast while consuming the CO2 and organic acids released by the yeast cells. Further, yeast can break complex sugars in the medium, which can then be utilized by microalgae thereby opening new options for copious and low-cost feedstocks such as agricultural residues. The current review provides a historical and technical overview of the existing studies on co-culturing of yeast and microalgae and elucidates the crucial factors that affect the symbiotic relationship between these two organisms. Furthermore, the review also highlighted the advantages and the future perspectives for paving a path towards a sustainable biodiesel product.

Delineating the molecular responses of a halotolerant microalga using integrated omics approach to identify genetic engineering targets for enhanced TAG production.

BACKGROUND: Harnessing the halotolerant characteristics of microalgae provides a viable alternative for sustainable biomass and triacylglyceride (TAG) production. Scenedesmus sp. IITRIND2 is a fast growing fresh water microalga that has the capability to thrive in high saline environments. To understand the microalga's adaptability, we studied its physiological and metabolic flexibility by studying differential protein, metabolite and lipid expression profiles using metabolomics, proteomics, real-time polymerase chain reaction, and lipidomics under high salinity conditions. RESULTS: On exposure to salinity, the microalga rewired its cellular reserves and ultrastructure, restricted the ions channels, and modulated its surface potential along with secretion of extrapolysaccharide to maintain homeostasis and resolve the cellular damage. The algal-omics studies suggested a well-organized salinity-driven metabolic adjustment by the microalga starting from increasing the negatively charged lipids, up regulation of proline and sugars accumulation, followed by direction of carbon and energy flux towards TAG synthesis. Furthermore, the omics studies indicated both de-novo and lipid cycling pathways at work for increasing the overall TAG accumulation inside the microalgal cells. CONCLUSION: The salt response observed here is unique and is different from the well-known halotolerant microalga; Dunaliella salina, implying diversity in algal response with species. Based on the integrated algal-omics studies, four potential genetic targets belonging to two different metabolic pathways (salt tolerance and lipid production) were identified, which can be further tested in non-halotolerant algal strains.

A novel rapid ultrasonication-microwave treatment for total lipid extraction from wet oleaginous yeast biomass for sustainable biodiesel production.

Oleaginous yeasts have emerged as a sustainable source of renewable oils for liquid biofuels. However, biodiesel production from them has a few constraints with respect to their cell disruption and lipid extraction techniques. The lipid extraction from oleaginous yeasts commonly includes dewatering and drying of cell biomass, which requires energy and time. The aim of this work was to establish a process for the lipid extraction from wet biomass applying acid catalyzed hot water, as well as microwave, and rapid ultrasonication-microwave treatment together with the conventional Bligh and Dyer method. In the wake of testing all procedures, it was revealed that rapid ultrasonication-microwave treatment has great potential to give high lipid content (70.86% w/w) on the cell dry weight basis. The lipid profile after treatment showed the presence of appropriate quantities of saturated (10.39 ±â€¯0.15%), monounsaturated (76.55 ±â€¯0.19%) and polyunsaturated fatty acids (11.49 ±â€¯0.23%) which further improves biodiesel quality compared to the other methods. To the best of our knowledge, this is the first report of using rapid ultrasonication-microwave treatment for the lipid extraction from wet oleaginous yeast biomass in the literature.

NMR-Based Metabolomic Approach To Elucidate the Differential Cellular Responses during Mitigation of Arsenic(III, V) in a Green Microalga.

Nuclear magnetic resonance (NMR)-based metabolomic approach is a high-throughput fingerprinting technique that allows a rapid snapshot of metabolites without any prior knowledge of the organism. To demonstrate the applicability of NMR-based metabolomics in the field of microalgal-based bioremediation, novel freshwater microalga Scenedesmus sp. IITRIND2 that showed hypertolerance to As(III, V) was chosen for evaluating the metabolic perturbations during arsenic stress in both its oxidation states As(III) and As(V). Using NMR spectroscopy, we were able to identify and quantify an array of ∼45 metabolites, including amino acids, sugars, organic acids, phosphagens, osmolytes, nucleotides, etc. The NMR metabolomic experiments were complemented with various biophysical techniques to establish that the microalga tolerated the arsenic stress using a complex interplay of metabolites. The two different arsenic states distinctly influenced the microalgal cellular mechanisms due to their altered physicochemical properties. Eighteen differentially identified metabolites related to bioremediation of arsenic were then correlated to the major metabolic pathways to delineate the variable stress responses of microalga in the presence of As(III, V).

Potential of aquatic oomycete as a novel feedstock for microbial oil grown on waste sugarcane bagasse.

Biodiesel production from vegetable oils is not sustainable and economical due to the food crisis worldwide. The development of a cost-effective non-edible feedstock is essential. In this study, we proposed to use aquatic oomycetes for microbial oils, which are cellulolytic fungus-like filamentous eukaryotic microorganisms, commonly known as water molds. They differ from true fungi as cellulose is present in their cell wall and chitin is absent. They show parasitic as well as saprophytic nature and have great potential to utilize decaying animal and plant debris in freshwater habitats. To study the triacylglycerol (TAG) accumulation in the aquatic oomycetes, the isolated water mold Achlya diffusa was cultivated under semi-solid-state conditions on waste sugarcane bagasse, which was compared with the cultivation in Czapek (DOX) medium. A. diffusa grown on waste sugarcane bagasse showed large lipid droplets in its cellular compartment and synthesized 124.03 ± 1.93 mg/gds cell dry weight with 50.26 ± 1.76% w/w lipid content. The cell dry weight and lipid content of this water mold decreased to 89.54 ± 1.21 mg/gds and 38.82% w/w, respectively, when cultivated on standard medium Czapek-Dox agar (CDA). For the fatty acid profile of A. diffusa grown in sugarcane bagasse and CDA, in situ transesterification (IST) and indirect transesterification (IDT) approaches were evaluated. The lipid profile of this mold revealed the presence of C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, C20:0, and C21:0 fatty acids, which is similar to vegetable oils. The biodiesel properties of the lipids obtained from A. diffusa satisfied the limits as determined by international standards ASTM-D6751 and EN-14214 demonstrating its suitability as a fuel for diesel engines.

Amaranth seeds (Amaranthus palmeri L.) as novel feedstock for biodiesel production by oleaginous yeast.

The potential of lipid accumulation by oleaginous yeast Cryptococcus vishniaccii grown on amaranth seed aqueous extract (AAE) media was assessed. Maximum cell biomass productivity of 104 mg/L/h, lipid productivity of 54 mg/L/h, and lipid content of 52.31% were recorded on AAE when carbon to nitrogen (C:N) ratio increased from 134 to 147 after removal of ammonia nitrogen. The lipid droplet (LD) size (2.32 ± 0.38 µm) was visualized by fluorescence microscopy using Nile red stain indicating maximum accumulated triacylglycerol (TAG) at C:N 147. Fatty acid methyl ester (FAME) profile obtained after transesterification of extracted lipid revealed the presence of palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2). Data showed the presence of high monounsaturated fatty acid (MUFA) content (68.17%) depicting improved winter operating conditions of biodiesel. Various quality parameters of biodiesel were evaluated and compared to the American and European biodiesel standards specifications. Based on the lipid productivity, distribution of fatty acids, and evaluated properties obtained; the lipid accumulation by C. vishniaccii utilizing amaranth seeds as substrate could serve as a feasible feedstock for biodiesel production.

Accelerated in vivo wound healing evaluation of microbial glycolipid containing ointment as a transdermal substitute.

A potent biosurfactant (BS) producing Bacillus licheniformis SV1 (NCBI GenBank Accession No. KX130852) was isolated from oil contaminated soil sample. Physicochemical investigations (TLC, HPLC, FTIR, GC-MS and NMR) revealed it to be glycolipid in nature. Fibroblast culture assay showed cytocompatibility and increased cell proliferation of 3T3/NIH fibroblast cells treated with this biosurfactant when checked using MTT assay and DAPI fluorescent staining. To evaluate the wound healing potential, BS ointment was formulated and checked for its spreadability and viscosity consistency. In vivo wound healing examination of full thickness skin excision wound rat model demonstrated the prompt re-epithelialization and fibroblast cell proliferation in the early phase while quicker collagen deposition in later phases of wound healing when BS ointment was used. These results validated the potential usage of BS ointment as a transdermal substitute for faster healing of impaired skin wound. Biochemical evaluation also substantiated the highest concentration of hydroxyproline (32.18±0.46, p<0.001) in the BS ointment treated animal tissue samples compared to the control. Hematoxylin-Eosin (H&E) and Masson's Trichrome staining validated the presence of increased amount of collagen fibers and blood vessels in the test animals treated with BS ointment.

Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast.

Phenol is reported to be one of the most toxic environmental pollutants present in the discharge of various industrial effluents causing a serious threat to the existing biome. Biodegradation of phenol by oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 was found to degrade 1000mg/l phenol. The pathways for phenol degradation by both ortho and meta-cleavage were proposed by the identification of metabolites and enzymatic assays of ring cleavage enzymes in the cell extracts. Results suggest that this oleaginous yeast degrade phenol via meta-cleavage pathway and accumulates a high quantity of lipid content (64.92%; wt/wt) as compared to control glucose synthetic medium (GSM). Meta-cleavage pathway of phenol degradation leads to formation of pyruvate and acetaldehyde. Both these end products feed as precursors for de novo triacylglycerols (TAG) biosynthesis pathway which causes accumulation of TAG in the lipid droplets (LD) of 6.12±0.78µm grown on phenol while 2.38±0.52µm observed on GSM. This was confirmed by fluorescence microscopic images of BODIPY505-515nm stained live yeast cells. GC-MS analysis of extracted total lipid showed enhanced amount of monounsaturated fatty acid (MUFA) which was as 51.87%, 58.33% and 62.98% in presence of 0.5, 0.75 and 1g/l of phenol.

Augmented lipid accumulation in ethyl methyl sulphonate mutants of oleaginous microalga for biodiesel production.

The aim of this work was to generate high lipid accumulating mutants of Chlorella minutissima (CM) using ethyl methyl sulphonate (EMS) as a random chemical mutagen. Amid the 5% surviving cells after exposure to EMS (2M), three fast growing mutants (CM2, CM5, CM7) were selected and compared with wild type for lipid productivity and biochemical composition. Among these mutants, CM7 showed the maximum biomass (2.4g/L) and lipid content (42%) as compared to wild type (1.5g/L; 27%). Further, the mutant showed high photosynthetic pigments with low starch content signifying the re-allocation of carbon flux to lipid. The obtained mutant showed no visible morphological changes in comparison to its WT. The fatty acid profile showed increase in monounsaturated fatty acids while decreased saturated and polyunsaturated fatty acids signifying good quality biodiesel. The mutant strain thus obtained can be optimized further and applied for enhanced biodiesel production.

Bioremediation of domestic and industrial wastewaters integrated with enhanced biodiesel production using novel oleaginous microalgae.

The study illustrates the synergistic potential of novel microalgal, Chlamydomonas debaryana IITRIND3, for phycoremediation of domestic, sewage, paper mill and dairy wastewaters and then subsequent utilisation of its biomass for biodiesel production. Among these wastewaters, maximum lipid productivity (87.5 ± 2.3 mg L-1 day-1) was obtained in dairy wastewater with removal efficiency of total nitrogen, total phosphorous, chemical oxygen demand and total organic carbon to be 87.56, 82.17, 78.57 and 85.97 %, respectively. Metal ions such as sodium, calcium, potassium and magnesium were also removed efficiently from the wastewaters tested. Pigment analysis revealed loss of chlorophyll a while increase in carotenoid content in algal cells cultivated in different wastewaters. Biochemical data of microalgae grown in different wastewaters showed reduction in protein content with an increase in carbohydrate and lipid contents. The major fatty acids in algal cells grown in dairy wastewater were C14:0, C16:0, C16:1, C18:0, C18:2 and C18:3. The physical properties of biodiesel derived from microalgae grown in dairy wastewater were in compliance with the ASTM D6751 and EN 14214 fuel standards and were comparable to plant oil methyl esters.

Recycled de-Oiled Algal Biomass Extract as a Feedstock for Boosting Biodiesel Production from Chlorella minutissima.

The investigation for the first time assesses the efficacy of recycled de-oiled algal biomass extract (DABE) as a cultivation media to boost lipid productivity in Chlorella minutissima and its comparison with Bold's basal media (BBM) used as control. Presence of organic carbon (3.8 ± 0.8 g/l) in recycled DABE resulted in rapid growth with twofold increase in biomass productivity as compared to BBM. These cells expressed four folds higher lipid productivity (126 ± 5.54 mg/l/d) as compared to BBM. Cells cultivated in recycled DABE showed large sized lipid droplets accumulating 54.12 % of lipid content. Decrement in carbohydrate (17.76 %) and protein content (28.12 %) with loss of photosynthetic pigments compared to BBM grown cells were also recorded. The fatty acid profiles of cells cultivated in recycled DABE revealed the dominance of C16:0 (39.66 %), C18:1 (29.41 %) and C18:0 (15.82 %), respectively. This model is self-sustained and aims at neutralizing excessive feedstock consumption by exploiting recycled de-oiled algal biomass for cultivation of microalgae, making the process cost effective.

Impact of oxidative and osmotic stresses on Candida albicans biofilm formation.

Candida albicans possesses an ability to grow under different host-driven stress conditions by developing robust protective mechanisms. In this investigation the focus was on the impact of osmotic (2M NaCl) and oxidative (5 mM H2O2) stress conditions during C. albicans biofilm formation. Oxidative stress enhanced extracellular DNA secretion into the biofilm matrix, increased the chitin level, and reduced virulence factors, namely phospholipase and proteinase activity, while osmotic stress mainly increased extracellular proteinase and decreased phospholipase activity. Fourier transform infrared and nuclear magnetic resonance spectroscopy analysis of mannan isolated from the C. albicans biofilm cell wall revealed a decrease in mannan content and reduced ß-linked mannose moieties under stress conditions. The results demonstrate that C. albicans adapts to oxidative and osmotic stress conditions by inducing biofilm formation with a rich exopolymeric matrix, modulating virulence factors as well as the cell wall composition for its survival in different host niches.

Boosting TAG Accumulation with Improved Biodiesel Production from Novel Oleaginous Microalgae Scenedesmus sp. IITRIND2 Utilizing Waste Sugarcane Bagasse Aqueous Extract (SBAE).

This investigation utilized sugarcane bagasse aqueous extract (SBAE), a nontoxic, cost-effective medium to boost triacylglycerol (TAG) accumulation in novel fresh water microalgal isolate Scenedesmus sp. IITRIND2. Maximum lipid productivity of 112 ± 5.2 mg/L/day was recorded in microalgae grown in SBAE compared to modified BBM (26 ± 3 %). Carotenoid to chlorophyll ratio was 12.5 ± 2 % higher than in photoautotrophic control, indicating an increase in photosystem II activity, thereby increasing growth rate. Fatty acid methyl ester (FAME) profile revealed presence of C14:0 (2.29 %), C16:0 (15.99 %), C16:2 (4.05 %), C18:0 (3.41 %), C18:1 (41.55 %), C18:2 (12.41), and C20:0 (1.21 %) as the major fatty acids. Cetane number (64.03), cold filter plugging property (-1.05 °C), and oxidative stability (12.03 h) indicated quality biodiesel abiding by ASTM D6751 and EN 14214 fuel standards. Results consolidate the candidature of novel freshwater microalgal isolate Scenedesmus sp. IITRIND2 cultivated in SBAE, aqueous extract made from copious, agricultural waste sugarcane bagasse to increase the lipid productivity, and could further be utilized for cost-effective biodiesel production.

Drug functionalized microbial polysaccharide based nanofibers as transdermal substitute.

In order to promote the natural healing process, drug-functionalized nanofibrous transdermal substitute was fabricated using gellan as chief polymer and polyvinyl alcohol (PVA) as supporting polymer via electrospinning technique. These fabricated nanofibers physiochemically mimic the extracellular matrix (ECM) which supports the cell growth. For neo-tissue regeneration in a sterilized environment, amoxicillin (Amx) was entrapped within these nanofibers. Entrapment of Amx in the nanofibers was confirmed by FESEM, FTIR, XRD and TG analysis. In vitro cell culture studies revealed that the fabricated non-cytotoxic nanofibers promoted enhance cell adherence and proliferation of human keratinocytes. A preliminary in vivo study performed on rat model for full thickness skin excision wound demonstrated the prompt re-epithelialization in early phase and quicker collagen deposition in later phases of wound healing in case of Amx-functionalized gellan/PVA nanofibers. Data collectively confirmed the potential usage of gellan based electrospun nanofibers as transdermal substitute for faster skin restoration.

Synergistic dynamics of nitrogen and phosphorous influences lipid productivity in Chlorella minutissima for biodiesel production.

The study synergistically optimized nitrogen and phosphorous concentrations for attainment of maximum lipid productivity in Chlorella minutissima. Nitrogen and phosphorous limited cells (N(L)P(L)) showed maximum lipid productivity (49.1±0.41mg/L/d), 1.47 folds higher than control. Nitrogen depletion resulted in reduced cell size with large sized lipid droplets encompassing most of the intracellular space while discrete lipid bodies were observed under nitrogen sufficiency. Synergistic N/P starvations showed more prominent effect on photosynthetic pigments as to individual deprivations. Phosphorous deficiency along with N starvation exhibited 17.12% decline in carbohydrate while no change in nitrogen sufficient cells were recorded. The optimum N(L)P(L) concentration showed balance between biomass and lipid by maintaining intermediate cell size, pigments, carbohydrate and proteins. FAME profile showed C14-C18 carbon chains in N(L)P(L) cells with biodiesel properties comparable to plant oil methyl esters. Hence, synergistic N/P limitation was effective for enhancing lipid productivity with reduced consumption of nutrients.

Modulation of Candida albicans Biofilm by Different Carbon Sources.

In the present investigation, the role of carbon sources (glucose, lactate, sucrose, and arabinose) on Candida albicans biofilm development and virulence factors was studied on polystyrene microtiter plates. Besides this, structural changes in cell wall component ß-glucan in presence of different carbon sources have also been highlighted. Biofilm formation was analyzed by XTT (2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay. Glucose-grown cells exhibited the highest metabolic activity during adhesion among all carbon sources tested (p < 0.05). However, cells exposed to sucrose exhibited highest biofilm formation and matrix polysaccharides secretion after 48 h. The results also correlated with the biofilm height and roughness measurements by atomic force microscopy. Exposure to lactate induced hyphal structures with the highest proteinase activity while arabinose-grown cells formed pseudohyphal structures possessing the highest phospholipase activity. Structural changes in ß-glucan characterized by Fourier transform infrared (FTIR) spectroscopy displayed characteristic band of ß-glucan at 892 cm(-1) in all carbon sources tested. The ß(1→6) to ß(1→3) glucan ratio calculated as per the band area of the peak was less in lactate (1.15) as compared to glucose (1.73), sucrose (1.62), and arabinose (2.85). These results signify that carbon sources influence C. albicans biofilm development and modulate virulence factors and structural organization of cell wall component ß-glucan.

A novel gellan-PVA nanofibrous scaffold for skin tissue regeneration: Fabrication and characterization.

In this investigation, we have introduced novel electrospun gellan based nanofibers as a hydrophilic scaffolding material for skin tissue regeneration. These nanofibers were fabricated using a blend mixture of gellan with polyvinyl alcohol (PVA). PVA reduced the repulsive force of resulting solution and lead to formation of uniform fibers with improved nanostructure. Field emission scanning electron microscopy (FESEM) confirmed the average diameter of nanofibers down to 50 nm. The infrared spectra (IR), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis evaluated the crosslinking, thermal stability and highly crystalline nature of gellan-PVA nanofibers, respectively. Furthermore, the cell culture studies using human dermal fibroblast (3T3L1) cells established that these gellan based nanofibrous scaffold could induce improved cell adhesion and enhanced cell growth than conventionally proposed gellan based hydrogels and dry films. Importantly, the nanofibrous scaffold are biodegradable and could be potentially used as a temporary substrate/or biomedical graft to induce skin tissue regeneration.

Biodiesel production from non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp using oleaginous yeast Rhodosporidium kratochvilovae HIMPA1.

This study explored biodiesel production from a low cost, abundant, non-edible lignocellulosic biomass from aqueous extract of Cassia fistula L. (CAE) fruit pulp. The CAE was utilized as substrate for cultivating novel oleaginous yeast Rhodosporidium kratochvilovae HIMPA1. This oleaginous yeast accumulates high amount of triacylglycerides as large intracellular lipid droplets (4.35±0.54µm) using CAE as sole nutritional source. Total lipids (4.86±0.54g/l) with lipid content of 53.18% (w/w) were produced by R. kratochvilovae HIMPA1 on CAE. The FAME profile obtained revealed palmitic acid (C16:0) 43.06%, stearic acid (C18:0) 28.74%, and oleic acid (C18:1) 17.34% as major fatty acids. High saturated fatty acids content (72.58%) can be blended with high PUFA feedstocks to make it an industrially viable renewable energy product.

Synergistic effect of fermentable and non-fermentable carbon sources enhances TAG accumulation in oleaginous yeast Rhodosporidium kratochvilovae HIMPA1.

Novel strategy for enhancing TAG accumulation by simultaneous utilization of fermentable and non-fermentable carbon sources as substrate for cultivation of oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 were undertaken in this investigation. The yeast strain showed direct correlation between the size of lipid bodies, visualized by BODIPY stain (493-515 nm) and TAG accumulation when examined on individual fermenting and non-fermenting carbon sources and their mixtures. Maximum TAG accumulation (µm) in glucose (2.38 ± 0.52), fructose (4.03 ± 0.38), sucrose (4.24 ± 0.45), glycerol (4.35 ± 0.54), xylulose (3.94 ± 0.12), and arabinose (2.98 ± 0.43) were observed. Synergistic effect of the above carbon sources (fermentable and non-fermentable) in equimolar concentration revealed maximum lipid droplet size of 5.35 ± 0.76 µm and cell size of 6.89 ± 0.97 µm. Total lipid content observed in mixed carbon sources was 9.26 g/l compared to glucose (6.2g/l). FAME profile revealed enhanced longer chain (C14:0-C24:0) fatty acids in mix carbon sources.