A comparative appraisal of three important oil yielding plants for their biodiesel potential.

In the present scenario, alternative energy sources are required to achieve the future economic prosperity where shortage of fossil fuels will be a limiting factor and hamper the global economic growth. Therefore, interest in biofuel is increasing continuously. The best way of sustainable development is fossil fuel supplementation with biodiesel to reduce the fossil fuel demand. Biodiesel is a clean burning, ester-based, oxygenated fuel derived from natural and renewable sources. Till now, majority of the people have worked on the biodiesel derived from edible oil. Instead of using edible oil, non-edible oil needs to be explored as feedstock for biofuel because half of the world's population is unable to afford the food oil as feedstock for fuel production. Looking at the significance of biodiesel and the resources of biofuel, in this paper, a comparative exhaustive study has been reported with for three important plants, namely Jatropha curcas, Pongemia pinnata and Balanites aegyptiaca. These plants were selected based on their biodiesel potential, availability, cultivation practices and general information available. The present study involves scientometric publications, comparison of fatty acid composition and biodiesel parameters. We have also compared climatic conditions for the growth of the plants, economic feasibility of biodiesel production and other ecological services. The study paves a way for sustainable solution to policy makers and foresters looking for selection of plant species as bioenergy resource.

Development of Robust Yeast Strains for Lignocellulosic Biorefineries Based on Genome-Wide Studies.

Lignocellulosic biomass has been widely studied as the renewable feedstock for the production of biofuels and biochemicals. Budding yeast Saccharomyces cerevisiae is commonly used as a cell factory for bioconversion of lignocellulosic biomass. However, economic bioproduction using fermentable sugars released from lignocellulosic feedstocks is still challenging. Due to impaired cell viability and fermentation performance by various inhibitors that are present in the cellulosic hydrolysates, robust yeast strains resistant to various stress environments are highly desired. Here, we summarize recent progress on yeast strain development for the production of biofuels and biochemical using lignocellulosic biomass. Genome-wide studies which have contributed to the elucidation of mechanisms of yeast stress tolerance are reviewed. Key gene targets recently identified based on multiomics analysis such as transcriptomic, proteomic, and metabolomics studies are summarized. Physiological genomic studies based on zinc sulfate supplementation are highlighted, and novel zinc-responsive genes involved in yeast stress tolerance are focused. The dependence of host genetic background of yeast stress tolerance and roles of histones and their modifications are emphasized. The development of robust yeast strains based on multiomics analysis benefits economic bioconversion of lignocellulosic biomass.

Bio-Based Compounds from Grape Seeds: A Biorefinery Approach.

Food and agricultural waste represents a growing problem with negative effects on the economy, environment, and human health. Winemaking produces byproducts with high added value, which can be used for new productions in several application fields. From the perspective of biorefinery and circular economy, grape seeds could be exploited by extracting bioactive compounds with high added value before using biomass for energy purposes. The markets concerned are, in addition to the food, cosmetics, and pharmaceuticals sectors, which use bioactive compounds, the sector of biopolymeric materials and of energy for the production of biohydrogen and biomethane. Generally, bioactive components should be investigated through an integrated and multidisciplinary study approach based on emerging analytical techniques; in this context, attention is addressed towards green and sustainable procedures; an update of extraction techniques, innovative technologies, and chemometrics are described. Nowadays, processes so far tested on a pilot scale for grape waste are developed to enhance the extraction yields. Here, a picture of the Italian experience applied to the byproducts of the wine industry is given.

Lipase Immobilization on Silica Xerogel Treated with Protic Ionic Liquid and its Application in Biodiesel Production from Different Oils.

Treated silica xerogel with protic ionic liquid (PIL) and bifunctional agents (glutaraldehyde and epichlorohydrin) is a novel support strategy used in the effective immobilization of lipase from Burkholderia cepacia (LBC) by covalent binding. As biocatalysts with the highest activity recovery yields, LBC immobilized by covalent binding with epichlorohydrin without (203%) and with PIL (250%), was assessed by the following the hydrolysis reaction of olive oil and characterized biochemically (Michaelis⁻Menten constant, optimum pH and temperature, and operational stability). Further, the potential transesterification activity for three substrates: sunflower, soybean, and colza oils, was also determined, achieving a conversion of ethyl esters between 70 and 98%. The supports and the immobilized lipase systems were characterized using Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), elemental analysis, and thermogravimetric (TG) analysis.

High-level expression and characterization of solvent-tolerant lipase.

In this study, the coding sequence of the lipase from Proteus sp. SW1 was optimized via codon optimization and subjected to expression in Pichia pastoris GS115. The maximum enzyme yield was 387 mg/L in the supernatants of the shake-flask culture. The purified recombinant lipase exhibited a specific activity of 130 U/mg toward p-nitrophenyl Laurate. Its optimum pH and temperature were 8.0 and 40°C, respectively. It was highly stable and even activated in water-miscible solvents, showing over 102% residual activity after 24 h incubation in ethanol, acetone, isopropanol and acetonitrile. In addition, the enzyme showed promoted activity with the increasing concentrations of methanol/ethanol and exhibited the maximum activity at 80%. In a solvent-free system for biodiesel synthesis with a one-step addition of methanol, the recombinant lipase displayed a 87% conversion rate toward palm oil at the high water content of 80%. The highly improved expression level and activity of the recombinant lipase may contribute to enable its commercial-scale production, and the unique properties would make it a particularly promising biocatalyst for biodiesel production in the future.

Screening of natural yeast isolates under the effects of stresses associated with second-generation biofuel production.

Robust microorganisms are required for sustainable second-generation biofuel production. We evaluated the growth and fermentation performance of six natural isolates that were derived from grape wine and medicinal herbs using a wide range of carbon sources, rice and wheat straw hydrolysates as well as stress conditions associated with second-generation ethanol production. Sequence analysis of the 5.8S internal transcribed spacer (ITS) and species-specific PCR amplification of the HO gene region assigned the natural isolates to Saccharomyces cerevisiae. Restriction fragment length polymorphism (RFLP) analysis of the mitochondrial DNA revealed that natural yeast isolates are genetically closer to the laboratory strain BY4741 than to the CEN.PK strains. Dextrose fermentation by a natural isolate, MTCC4780, under semi-anaerobic conditions produced maximum ethanol yields of 0.44 g/g and 0.39 g/g, respectively, with and without the stresses encountered during lignocellulosic ethanol fermentation. However, MTCC4780 produced ethanol yields of 0.48 g/g, 0.42 g/g and 0.45 g/g, respectively, with glucose, rice and wheat straw enzymatic hydrolysate fermentation in a bioreactor. The isolates MTCC4781 and MTCC4796 showed higher growth and fermentation performance than did MTCC4780 in the presence of elevated temperature and pre-treatment inhibitors. Taken together, the MTCC4780, MTCC4781 and MTCC4796 strains have the potential to serve as a platform for lignocellulosic ethanol production under stresses associated with second-generation biofuel production.

Production of biodiesel from vegetable oil and microalgae by fatty acid extraction and enzymatic esterification.

The aim of this work was to obtain biodiesel (methyl esters) from the saponifiable lipids (SLs) fraction of the microalga Nannochloropsis gaditana, whose biomass dry weight contains 12.1 wt% of these lipids. SLs were extracted from the microalga as free fatty acids (FFAs) for subsequent transformation to methyl esters (biodiesel) by enzymatic esterification. Extraction as FFAs rather than as SLs allows them to be obtained with higher purity. Microalgal FFAs were obtained by direct saponification of lipids in the biomass and subsequent extraction-purification with hexane. Esterification of FFAs with methanol was catalysed by lipase Novozym 435 from Candida antarctica. Stability studies of this lipase in the operational conditions showed that the esterification degree (ED) attained with the same batch of lipase remained constant over six reaction cycles (36 h total reaction time). The optimal conditions attained for 4 g of FFAs were 25°C, 200 rpm, methanol/FFA molar ratio of 1.5:1, Novozym 435/FFA ratio of 0.025:1 w/w and 4 h reaction time. In these conditions the ED attained was 92.6%, producing a biodiesel with 83 wt% purity from microalgal FFAs. Several experimental scales were tested (from 4 to 40 g FFAs), and in all cases similar EDs were obtained.

Immobilization of Pseudomonas fluorescens lipase on hydrophobic supports and application in biodiesel synthesis by transesterification of vegetable oils in solvent-free systems.

This work describes the preparation of biocatalysts for ethanolysis of soybean and babassu oils in solvent-free systems. Polystyrene, Amberlite (XAD-7HP), and octyl-silica were tested as supports for the immobilization of Pseudomonas fluorescens lipase (PFL). The use of octyl-silica resulted in a biocatalyst with high values of hydrolytic activity (650.0 ± 15.5 IU/g), immobilization yield (91.3 ± 0.3 %), and recovered activity (82.1 ± 1.5 %). PFL immobilized on octyl-silica was around 12-fold more stable than soluble PFL, at 45 °C and pH 8.0, in the presence of ethanol at 36 % (v/v). The biocatalyst provided high vegetable oil transesterification yields of around 97.5 % after 24 h of reaction using babassu oil and around 80 % after 48 h of reaction using soybean oil. The PFL-octyl-silica biocatalyst retained around 90 % of its initial activity after five cycles of transesterification of soybean oil. Octyl-silica is a promising support that can be used to immobilize PFL for subsequent application in biodiesel synthesis.

Carbon source utilization and inhibitor tolerance of 45 oleaginous yeast species.

Conversion of lignocellulosic hydrolysates to lipids using oleaginous (high lipid) yeasts requires alignment of the hydrolysate composition with the characteristics of the yeast strain, including ability to utilize certain nutrients, ability to grow independently of costly nutrients such as vitamins, and ability to tolerate inhibitors. Some combination of these characteristics may be present in wild strains. In this study, 48 oleaginous yeast strains belonging to 45 species were tested for ability to utilize carbon sources associated with lignocellulosic hydrolysates, tolerate inhibitors, and grow in medium without supplemented vitamins. Some well-studied oleaginous yeast species, as well as some that have not been frequently utilized in research or industrial production, emerged as promising candidates for industrial use due to ability to utilize many carbon sources, including Cryptococcus aureus, Cryptococcus laurentii, Hannaella aff. zeae, Tremella encephala, and Trichosporon coremiiforme. Other species excelled in inhibitor tolerance, including Candida aff. tropicalis, Cyberlindnera jadinii, Metschnikowia pulcherrima, Schwanniomyces occidentalis and Wickerhamomyces ciferrii. No yeast tested could utilize all carbon sources and tolerate all inhibitors tested. These results indicate that yeast strains should be selected based on characteristics compatible with the composition of the targeted hydrolysate. Other factors to consider include the production of valuable co-products such as carotenoids, availability of genetic tools, biosafety level, and flocculation of the yeast strain. The data generated in this study will aid in aligning yeasts with compatible hydrolysates for conversion of carbohydrates to lipids to be used for biofuels and other oleochemicals.

Solid-state fermentation of Jatropha seed cake for optimization of lipase, protease and detoxification of anti-nutrients in Jatropha seed cake using Aspergillus versicolor CJS-98.

This study focused on the solid-state fermentation of Jatropha seed cake (JSC), a byproduct generated after biodiesel production. Presence of anti-nutritional compounds and toxins restricts its application in livestock feed. The disposal of the JSC is a major environmental problem in the future, due to the generation of huge quantity of JSC after biodiesel extraction. Hence the JSC was assessed for its suitability as substrate for production and optimization of lipase and protease from Aspergillus versicolor CJS-98 by solid-state fermentation (SSF). The present study was also focused on the biodetoxification of anti-nutrients and toxins in JSC. The SSF parameters were optimized for maximum production of lipase and protease. Under the optimized conditions, the JSC supplemented with maltose and peptone (2%), adjusted to pH 7.0, moisture content 40%, inoculated with 1 × 10(7) spores per 5 g cake and incubated at 25°C, produced maximum lipase, 1288 U/g and protease, 3366 U/g at 96 h. The anti-nutrients like phytic acid (6.08%), tannins (0.37%), trypsin inhibitors (697.5 TIU/g), cyanogenic glucosides (692.5 µg/100 g), and lectins (0.309 mg/ml), were reduced to 1.70%, 0.23%, 12.5 TIU/g, 560.6 µg/100 g and 0.034 mg/ml respectively. The main toxic compound phorbol esters content in the JSC was reduced from 0.083% to 0.015% after SSF. Our results indicate that viability of SSF to utilize the huge amount of seed cake generated after extraction of biodiesel, for production of industrial enzymes and biodetoxification of anti-nutrients, toxins.

Dynamic processes of indigenous microorganisms from a low-temperature petroleum reservoir during nutrient stimulation.

Compared to medium-high temperature petroleum reservoirs (30°C-73°C), little is known about microbial regulation by nutrients in low-temperature reservoirs. In this study, we report the performance (oil emulsification and biogas production) and community structure of indigenous microorganisms from a low-temperature (22.6°C) petroleum reservoir during nutrient stimulation. Culture-dependent approaches indicated that the number of hydrocarbon-oxidizing bacteria (HOB), nitrate-reducing bacteria (NRB) and methane-producing bacteria (MPB) increased by between 10- and 1000-fold, while sulfate-reducing bacteria (SRB) were observed at low levels during stimulation. Phylogenetic analysis of the 16S rRNA gene indicated that Pseudomonas, Ochrobactrum, Acinetobacter, Halomonas and Marinobacter, which have the capability to produce surfactants, were selectively enriched. Methanoculleus, Methanosaeta, Methanocorpusculum and Methanocalculus showed the largest increase in relative abundance among archaea. Micro-emulsion formed with an average oil droplet diameter of 14.3 µm (ranging between 4.1 µm and 84.2 µm) during stimulation. Gas chromatographic analysis of gas production (186 mL gas/200 mL medium) showed the levels of CO2 and CH4 increased 8.97% and 6.21%, respectively. Similar to medium-high temperature reservoirs, HOB, NRB, SRB and MPB were ubiquitous in the low-temperature reservoir, and oil emulsification and gas production were the main phenomena observed during stimulation. Oil emulsification required a longer duration of time to occur in the low-temperature reservoir.

The genome of the recently domesticated crop plant sugar beet (Beta vulgaris).

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world's annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714-758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant's potential in energy biotechnology.

In situ self-catalyzed reactive extraction of germinated oilseed with short-chained dialkyl carbonates for biodiesel production.

In order to eliminate the expense associated with solvent extraction and oil cleanup, and reduce the processing steps in biodiesel production, reactive extraction has become a focus of research in recent years. In this study, germinated castor seed was used as substrate and catalyst, dimethyl carbonate (DMC) was used as acyl acceptor and oil extractant to produce biodiesel. The optimum conditions were as follows: the germination time of castor seed was 72 h, DMC/germinated seed ratio was 12.5 ml/g, reaction temperature was 35°C, and water content was 2.11%. The biodiesel yield could reach as much as 87.41% under the optimized conditions. This germinated oilseed self-catalyzed reactive extraction can be a promising route for biodiesel production.

Pollen baiting facilitates the isolation of marine thraustochytrids with potential in omega-3 and biodiesel production.

Marine heterotrophic microbes are capable of accumulating large amounts of lipids, omega-3 fatty acids, carotenoids, and have potential for biodiesel production. Pollen baiting using Pinus radiata pollen grain along with direct plating techniques were used in this study as techniques for the isolation of oil-producing marine thraustochytrid species from Queenscliff, Victoria, Australia. Thirteen isolates were obtained using either direct plating or using pine pollen, with pine pollen acting as a specific substrate for the surface attachment of thraustochytrids. The isolates obtained from the pollen baiting technique showed a wide range of docosahexaenoic acid (DHA) accumulation, from 11 to 41 % of total fatty acid content (TFA). Direct plating isolates showed a moderate range of DHA accumulation, from 19 to 25 % of TFA. Seven isolates were identified on the basis of 18S rRNA sequencing technique as Thraustochytrium species, Schizochytrium species, and Ulkenia species. Although both methods appear to result in the isolation of similar strains, pollen baiting proved to be a simpler method for the isolation of these relatively slow-growing organisms.

Biochemical composition of green alga Chlorella minutissima in mixotrophic cultures under the effect of different carbon sources.

Mixotrophic growth of Chlorella minutissima with carbon supplements such as glucose, glycerol, succinate, molasses and press mud resulted in maximum biomass accumulation in glucose supplemented culture. Lipid content was maximum with molasses followed by press mud, fatty acid compositions of which also were best suited for biodiesel production.

Microbial engineering for the production of advanced biofuels.

Advanced biofuels produced by microorganisms have similar properties to petroleum-based fuels, and can 'drop in' to the existing transportation infrastructure. However, producing these biofuels in yields high enough to be useful requires the engineering of the microorganism's metabolism. Such engineering is not based on just one specific feedstock or host organism. Data-driven and synthetic-biology approaches can be used to optimize both the host and pathways to maximize fuel production. Despite some success, challenges still need to be met to move advanced biofuels towards commercialization, and to compete with more conventional fuels.