Fueling the future; plant genetic engineering for sustainable biodiesel production

Biodiesel has huge potentials as a green and technologically feasible alternative to fossil diesel. However, biodiesel production from edible oil crops has been widely criticized while nonedible oil plants are associated with some serious disadvantages, such as high cost, low oil yield, and unsuitable oil composition. The next generation sequencing (NGS), omics technologies, and genetic engineering have opened new paths toward achieving high performance-oil plants varieties for commercial biodiesel production. The intent of the present review paper is to review and critically discuss the recent genetic and metabolic engineering strategies developed to overcome the shortcoming faced in nonedible plants, including Jatropha curcas and Camelina sativa, as emerging platforms for biodiesel production. These strategies have been looked into three different categories. Through the first strategy aimed at enhancing oil content, the key genes involved in triacylglycerols (TAGs) biosynthesis pathway (e.g., diacylglycerol acyltransferase (DGAT), acetyl-CoA carboxylase (ACCase), and glycerol‐3‐phosphate dehydrogenase (GPD1)), genes affecting seed size and plant growth (e.g., transcription factors (WRI1), auxin response factor 19 (ARF19),  leafy cotyledon1 (LEC1), purple acid phosphatase 2 (PAP2), G-protein c subunit 3 (AGG3), and flowering locus T (FT)), as well as genes involved in TAGs degradation (e.g., sugar-dependent protein 1 triacylglycerol lipase (SDP1)) have been deliberated. While through the second strategy targeting enhanced oil composition, suppression of the genes involved in the biosynthesis of linoleic acids (e.g., fatty acid desaturase (FAD2), fatty acid elongase (FAE1), acyl-ACP thioesterase (FATB), and ketoacyl-ACP synthase II (KASII)), suppression of the genes encoding toxic metabolites (curcin precursor and casbene synthase (JcCASA)), and finally, engineering the genes responsible for the production of unusual TAGs (e.g., Acetyl-TAGs and hydroxylated fatty acids (HFA)) have been debated. In addition to those, enhancing tolerance to biotic (pest and disease) and abiotic (drought, salinity, freezing, and heavy metals) stresses as another important genetic engineering strategy to facilitate the cultivation of nonedible oil plants under conditions unsuitable for food crops has been addressed. Finally, the challenges faced prior to successful commercialization of the resultant GM oil plants such have been presented

Study of Acid Hydrolysis on Organic Waste: Understanding The Effect of Delignification and Particle Size

Organic wastes from Swiettenia marcophylla L, Artocarpus heterophyllus L, Mangifera indica L, and Annona muricata L were prepared by grinding into 0.1875, 0.3750, 0.7500 mm of particle size and delignified by 2% NaOH at 80°C for 90 minutes. Acid dilution hydrolysis process with H2SO4 1% was performed at 150°C for 120 minutes in a closed reactor. The effect of particle size and delignification on and reducing sugar concentration were investigated. The result showed (1) leaves that can be used as raw material to produce hydrogen should have 38–49% cellulose and hemicellulose. (2) Reducing sugar concentration increased with particle size reduction and delignification. (3) the best result with the highest reducing sugar concentration was achieved by 0.1875 mm particle size with delignification on Annona muricata L

Harnessing the potential of ligninolytic enzymes for lignocellulosic biomass pretreatment

Abundant lignocellulosic biomass from various industries provides a great potential feedstock for the production of value-added products such as biofuel, animal feed, and paper pulping. However, low yield of sugar obtained from lignocellulosic hydrolysate is usually due to the presence of lignin that acts as a protective barrier for cellulose and thus restricts the accessibility of the enzyme to work on the cellulosic component. This review focuses on the significance of biological pretreatment specifically using ligninolytic enzymes as an alternative method apart from the conventional physical and chemical pretreatment. Different modes of biological pretreatment are discussed in this paper which is based on (i) fungal pretreatment where fungi mycelia colonise and directly attack the substrate by releasing ligninolytic enzymes and (ii) enzymatic pretreatment using ligninolytic enzymes to counter the drawbacks of fungal pretreatment. This review also discusses the important factors of biological pretreatment using ligninolytic enzymes such as nature of the lignocellulosic biomass, pH, temperature, presence of mediator, oxygen, and surfactant during the biodelignification process

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass : a comprehensive review

Recently, lignocellulosic biomass as the most abundant renewable resource has been widely considered for bioalcohols production. However, the complex structure of lignocelluloses requires a multi-step process which is costly and time consuming. Although, several bioprocessing approaches have been developed for pretreatment, saccharification and fermentation, bioalcohols production from lignocelluloses is still limited because of the economic infeasibility of these technologies. This cost constraint could be overcome by designing and constructing robust cellulolytic and bioalcohols producing microbes and by using them in a consolidated bioprocessing (CBP) system. This paper comprehensively reviews potentials, recent advances and challenges faced in CBP systems for efficient bioalcohols (ethanol and butanol) production from lignocellulosic and starchy biomass. The CBP strategies include using native single strains with cellulytic and alcohol production activities, microbial co-cultures containing both cellulytic and ethanologenic microorganisms, and genetic engineering of cellulytic microorganisms to be alcohol-producing or alcohol producing microorganisms to be cellulytic. Moreover, high-throughput techniques, such as metagenomics, metatranscriptomics, next generation sequencing and synthetic biology developed to explore novel microorganisms and powerful enzymes with high activity, thermostability and pH stability are also discussed. Currently, the CBP technology is in its infant stage, and ideal microorganisms and/or conditions at industrial scale are yet to be introduced. So, it is essential to bring into attention all barriers faced and take advantage of all the experiences gained to achieve a high-yield and low-cost CBP process

Optimization of biotechnological production of xylitol by a Candida tropicalis strain using Response Surface Methodology

Introduction: Xylitol is known as one of the most commonly used dietary sugars in food and pharmaceutical industries. The common methodology for production of xylitol is a chemical process which requires high energy and is costly. Biotechnological production of xylitol using microorganisms is an alternative process that is environmentally friendly and cost effective. So, the objective of the present study was to optimize biotechnological production of xylitol from xylose using Candida tropicalis NCIM 3119 strain in the framework of Central Composite Design (CCD) and Response Surface Methodology (RSM). Materials and methods: Four independent factors including temperature (27, 32 and 37°C), pH (3, 5 and 7), xylose concentration (30, 50 and 70 g/l) and yeast extract concentration (3, 7.5 and 12 g/l) were selected, and the xylitol yield (Yp/s= gram xylitol per gram xylose utilized) and biomass production were calculated. Results: Based on the constructed model, maximum expected xylitol yield (Yp/s= 0.73) was achieved when temperature, pH, and xylose and yeast extract concentrations were 32.7°C, 4.7, 54.2 g/l and 12 g/l, respectively. To confirm the calculated model, an experiment for xylitol production by the strain in the optimum condition was designed at Erlenmeyer level. The results showed that observed xylitol yield and concentration and also biomass of the strain were 0.69, 36.7 g/l and 11.1 g/l, respectively, which were in accordance with the model. Discussion and conclusion: Based on the results, it could be concluded that the environmental parameters, including nitrogen source, temperature, pH and xylose and nitrogen source concentrations were optimized to enhance biotechnological production of xylitol, and the final concentration of 36.7 g/l xylitol with 0.69 yield efficiency was achieved

Fusarium culmorum affects expression of biofilm formation key genes in Bacillus subtilis

Abstract It is known that there is correlation between biofilm formation and antagonistic activities of Bacillus subtilis strains; but, the mechanism of this correlation is not clear. So, the effect of the plant pathogen (Fusarium culmorum) on the biofilm formation in a B. subtilis strain with high antagonistic and biofilm formation activities was studied. The expression of sinR and tasA genes involved in the biofilm formation was studied in both single culture of bacterium (B) and co-culture with F. culmorum (FB) using real-time PCR. The results revealed that the expression of the sinR gene in both B and FB conditions was continuously decreased during the biofilm formation period and, after 24 h (B4 and FB4), it reached 1% and 0.3% at the planktonic phase (B1), respectively, whereas the expression of the tasA was continuously increased and was 5.27 and 30 times more than that at the planktonic phase (B1) after 24 h, respectively. So, the expression reduction rate for sinR (3 times) and the expression increasing rate for tasA (6 times) were significantly higher in FB conditions than the B ones. The relative expression of sinR in FB1 (planktonic phase), FB2 (8 h), FB3(12 h), and FB4 (24 h) times was 0.65, 0.44, 0.35, and 0.29, whereas the tasA gene expression was 2.98, 3.44, 4.37, and 5.63-fold of the one at coordinate time points in B conditions, respectively. The significant expression reduction of sinR and increase of tasA confirmed that the presence of pathogen could stimulate biofilm formation in the antagonistic bacterium

Molecular characterization and transient expression in plants of a Mirabilis jalapa antiviral protein (MAP), and its use in functional studies

33 p.-8 fig.Crude protein fractions from Mirabilis jalapa of northern Iran containing Mirabilis Antiviral Protein (MAP) reduced the systemic accumulation of a plant RNA virus in Nicotiana benthamiana. A MAP gene was amplified by RT-PCR from total RNAs isolated from these Iranian M. jalapa plants (MAP-Tehran) without the 5´-end signal peptide sequence, to set up an experimental system that would transiently express the protein in the cell cytosol by agroinfiltration and simultaneously trigger a response to study its antiviral effects. The amplified fragment was first cloned into a high-copy plasmid without promoters flanking the cloning site and its nucleotide and amino acid sequences were determined and compared to available MAP sequences. Two putative ribosome inhibitor domains were found in the protein N-terminal and C-terminal regions, respectively. The cloned MAP-Tehran was transferred to binary vectors for agroinfiltration. Only constructs that expressed MAP-Tehran fused to the N- or C- halves of the monomeric red fluorescent protein used for bimolecular fluorescence complementation assays were obtained. In N. benthamiana leaves agroinfiltrated with these constructs, signs of cell plasmolysis became detectable under confocal microscopy at day 3 after infiltration (dai), and from day 4 necrotic lesions were visible. In agroinfiltrated patches at 2–3 dai, before the onset of cell death, reduced accumulation of a co-expressed reporter gene and also of a mechanically inoculated virus were observed. A double-alanine substitution (Glu → Ala, Arg → Ala) in the C-terminal region inhibitor domain of the MAP constructs sufficed to abolish all of these effects. Agroinfiltration of either the necrosis-inducing or non-inducing MAP variants caused no significant effect on the accumulation of an RNA virus inoculated on upper, non-infiltrated leaves.Peer reviewe