Biotechnology for Biofuels

Decreasing fossil fuels and its impact on global warming have led to an increasing demand for its replacement by sustainable renewable biofuels. Microalgae may offer a potential feedstock for renewable biofuels capable of converting atmospheric CO2 to substantial biomass and valuable biofuels, which is of great importance for the food and energy industries. Parachlorella kessleri, a marine unicellular green alga belonging to class Trebouxiophyceae, accumulates large amount of lipids under nutrient-deprived conditions. The present study aims to understand the metabolic imprints in order to elucidate the physiological mechanisms of lipid accumulations in this microalga under nutrient deprivation. Molecular profiles were obtained using gas chromatography–mass spectrometry (GC–MS) of P. kessleri subjected to nutrient deprivation. Relative quantities of more than 60 metabolites were systematically compared in all the three starvation conditions. Our results demonstrate that in lipid metabolism, the quantities of neutral lipids increased significantly followed by the decrease in other metabolites involved in photosynthesis, and nitrogen assimilation. Nitrogen starvation seems to trigger the triacylglycerol (TAG) accumulation rapidly, while the microalga seems to tolerate phosphorous limitation, hence increasing both biomass and lipid content. The metabolomic and lipidomic profiles have identified a few common metabolites such as citric acid and 2-ketoglutaric acid which play significant role in diverting flux towards acetyl-CoA leading to accumulation of neutral lipids, whereas other molecules such as trehalose involve in cell growth regulation, when subjected to nutrient deprivation. Understanding the entire system through qualitative (untargeted) metabolome approach in P. kessleri has led to identification of relevant metabolites involved in the biosynthesis and degradation of precursor molecules that may have potential for biofuel production, aiming towards the vision of tomorrow’s bioenergy needs.

The presence of soluble lignin, furfural and hydroxymethylfurfural (HMF) in industrial pre-hydrolysis liquor (PHL) from the pulping process can inhibit its bioconversion into bioethanol and other biochemicals. Although various technologies have been developed to remove these inhibitors, certain amounts of sugars are also inevitably removed during the treatment process. Hence, polystyrene divinylbenzene (PS-DVB) resin was used as an adsorptive material to simultaneously remove fermentation inhibitors while retaining sugars with high yields to improve the fermentability of PHL after acid hydrolysis by enriching its xylose concentration. The fermentability of acid-hydrolyzed PHL (A-PHL) was evaluated by the bioconversion into ethanol and xylosic acid (XA) after treatment with PS-DVB resin. The results showed that the highest xylose concentration (101.1 g/L) in PHL could be obtained by acid hydrolysis at 100 °C for 80 min with 4% acid, while the concentration of fermentation inhibitors (furfural, HMF and lignin) in PHL could also be significantly improved during the acid-hydrolysis process. After treatment with PS-DVB resin, not only were 97% of lignin, 92% of furfural, and 97% of HMF removed from A-PHL, but also 96% of xylose was retained for subsequent fermentation. With resin treatment, the fermentability of A-PHL could be improved by 162–282% for ethanol production from A-PHL containing 30–50 g/L xylose and by 18–828% for XA production from A-PHL containing 90–150 g/L xylose. These results confirmed that PS-DVB resin can remove inhibitors from PHL before producing value-added products by bioconversion. In addition, this work will ideally provide a concept for producing value-added chemicals from pre-hydrolysis liquor, which is regarded as the waste stream in the pulping process.

Syntrophic acetate oxidation (SAO) is the predominant pathway for methane production in high ammonia anaerobic digestion processes. The bacteria (SAOB) occupying this niche and the metabolic pathway are poorly understood. Phylogenetic diversity and strict cultivation requirements hinder comprehensive research and discovery of novel SAOB. Most SAOB characterised to date are affiliated to the physiological group of acetogens. Formyltetrahydrofolate synthetase is a key enzyme of both acetogenic and SAO metabolism. The encoding fhs gene has therefore been identified as a suitable functional marker, using a newly designed primer pair. In this comparative study, we used a combination of terminal restriction fragment length polymorphism profiling, clone-based comparison, qPCR and Illumina amplicon sequencing to assess the bacterial community and acetogenic sub-community prevailing in high- and low-ammonia laboratory-scale digesters in order to delineate potential SAOB communities. Potential candidates identified were further tracked in a number of low-ammonia and high-ammonia laboratory-scale and large-scale digesters in order to reveal a potential function in SAO. All methodical approaches revealed significant changes in the bacterial community composition concurrently with increasing ammonia and predominance of SAO. The acetogenic community under high ammonia conditions was revealed to be generally heterogeneous, but formed distinct phylogenetic clusters. The clusters differed clearly from those found under low-ammonia conditions and represented an acetogenic assemblage unique for biogas processes and recurring in a number of high-ammonia processes, indicating potential involvement in SAO. The phylogenetic affiliation and population dynamics observed point to a key community, belonging mainly to the Clostridia class, in particular to the orders Clostridiales and Thermoanaerobacterales, which appear to specialise in SAO rather than being metabolically versatile. Overall, the results reported here provide evidence of functional importance of the bacterial families identified in high-ammonia systems and extend existing knowledge of bacterial and acetogenic assemblages at low and high ammonia levels. This information will be of help in monitoring and assessing the impacts on the SAOB community in order to identify characteristics of robust and productive high ammonia biogas processes.

Environmental issues and shortage of fossil fuels have turned the public interest to the utilization of renewable, environmentally friendly fuels, such as ethanol. In order to minimize the competition between fuels and food production, researchers are focusing their efforts to the utilization of wastes and by-products as raw materials for the production of ethanol. household food wastes are being produced in great quantities in European Union and their handling can be a challenge. Moreover, their disposal can cause severe environmental issues (for example emission of greenhouse gasses). On the other hand, they contain significant amounts of sugars (both soluble and insoluble) and they can be used as raw material for the production of ethanol. Household food wastes were utilized as raw material for the production of ethanol at high dry material consistencies. A distinct liquefaction/saccharification step has been included to the process, which rapidly reduced the viscosity of the high solid content substrate, resulting in better mixing of the fermenting microorganism. This step had a positive effect in both ethanol production and productivity, leading to a significant increase in both values, which was up to 40.81% and 4.46 fold, respectively. Remaining solids (residue) after fermentation at 45% w/v dry material (which contained also the unhydrolyzed fraction of cellulose), were subjected to a hydrothermal pretreatment in order to be utilized as raw material for a subsequent ethanol fermentation. This led to an increase of 13.16% in the ethanol production levels achieving a final ethanol yield of 107.58 g/kg dry material. In conclusion, the ability of utilizing household food waste for the production of ethanol at elevated dry material content has been demonstrated. A separate liquefaction/saccharification process can increase both ethanol production and productivity. Finally, subsequent fermentation of the remaining solids could lead to an increase of the overall ethanol production yield.

Production of valuable metabolites by Yarrowia lipolytica using renewable raw materials is of major interest for sustainable food and energy. Galactose is a monosaccharide found in galactomannans, hemicelluloses, gums, and pectins. Yarrowia lipolytica was found to express all the Leloir pathway genes for galactose utilization, which encode fully functional proteins. Gene organization and regulation in Y. lipolytica resembles filamentous fungi rather than Saccharomyces cerevisiae. After Y. lipolytica was grown on mixture of glucose and galactose, it was then able to metabolize galactose, including when glucose concentrations were higher than 4 g/L. However, glucose was still the preferred carbon source. Nonetheless, a strain overexpressing the four ylGAL genes of the Leloir pathway was able to efficiently use galactose as its sole carbon source. This mutant was used to produce citric acid and lipids from galactose; the yields were comparable to or greater than that obtained for the parental strain (W29) on glucose. The construction of a Y. lipolytica strain able to produce citric acid and lipids from galactose is a very important step in bypassing issues related to the use of food-based substrates in industrial applications.

Plant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion into Arabidopsis cell walls (referred to here as FerEX). The transgenic Arabidopsis plants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants. This study demonstrated that extracellular expression of ferritin in Arabidopsis can produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.

Jatropha curcas L. (Jatropha) is a potential biodiesel crop that can be cultivated on marginal land because of its strong tolerance to drought and low soil nutrient content. However, seed yield remains low. To enhance the commercial viability and green index of Jatropha biofuel, a systemic and coordinated approach must be adopted to improve seed oil and biomass productivity. Here, we present our investigations on the Jatropha-associated nitrogen-fixing bacteria with an aim to understand and exploit the unique biology of this plant from the perspective of plant–microbe interactions. An analysis of 1017 endophytic bacterial isolates derived from different parts of Jatropha revealed that diazotrophs were abundant and diversely distributed into five classes belonging to α, β, γ-Proteobacteria, Actinobacteria and Firmicutes. Methylobacterium species accounted for 69.1 % of endophytic bacterial isolates in leaves and surprisingly, 30.2 % which were able to fix nitrogen that inhabit in leaves. Among the Methylobacterium isolates, strain L2-4 was characterized in detail. Phylogenetically, strain L2-4 is closely related to M. radiotolerans and showed strong molybdenum-iron dependent acetylene reduction (AR) activity in vitro and in planta. Foliar spray of L2-4 led to successful colonization on both leaf surface and in internal tissues of systemic leaves and significantly improved plant height, leaf number, chlorophyll content and stem volume. Importantly, seed production was improved by 222.2 and 96.3 % in plants potted in sterilized and non-sterilized soil, respectively. Seed yield increase was associated with an increase in female–male flower ratio. The ability of Methylobacterium to fix nitrogen and colonize leaf tissues serves as an important trait for Jatropha. This bacteria–plant interaction may significantly contribute to Jatropha’s tolerance to low soil nutrient content. Strain L2-4 opens a new possibility to improve plant’s nitrogen supply from the leaves and may be exploited to significantly improve the productivity and Green Index of Jatropha biofuel.

Cultivation of microalgae in wastewater could significantly contribute to wastewater treatment, biodiesel production, and thus the transition to renewable energy. However, more information on effects of environmental factors, including light intensity, on their growth and composition (particularly fatty acid contents) is required. Therefore, we investigated the biomass and fatty acid production of four microalgal species, isolated in the Northern hemisphere and grown at three light intensities (50, 150 and 300 μE m−2 s−1). Increases in light intensities resulted in higher biomass of all four species and, importantly, raised fatty acid contents of both Desmodesmus sp. and Scenedesmus obliquus. Fourier-transform IR spectrometry analysis showed that the increases in fatty acid content were associated with reductions in protein, but not carbohydrate, contents. Assessment of fatty acid composition revealed that increasing light intensity led to higher and lower contents of oleic (18:1) and linolenic (18:3) acids, respectively. The microalgae consumed more than 75% of the nitrogen and phosphorus present in the wastewater used as growth medium. The results show the importance of optimizing light intensities to improve fatty acid production by microalgae and their quality as sources of biodiesel. In addition, increase in fatty acid content is associated with decrease in protein content.