Renewable Bioenergy Production Research Articles

Enhanced furfural extraction using neoteric hydrophobic solvents for sustainable biomass recovery and bioenergy applications

The recovery of furfural from hemicellulosic biowastes is important for developing sustainable and renewable energy alternatives to fossil fuels. However, current methods are inefficient and environmentally questionable. To address this issue, this study introduces neoteric hydrophobic solvents, specifically deep eutectic solvents (DESs) and ionic liquids (ILs). Of the 32 solvents tested, thymol:decanoic acid 1:1 (Thy:DecA) DES and trihexyltetradecyl phosphonium bis(trifluoro methylsulfonyl) imide [P14,6,6,6][NTf2] IL were the most effective, with extraction efficiencies of 94.1% and 97.1%, respectively. These solvents outperformed the reference solvent toluene, with an efficiency of 81.2%, while also showing favorable characteristics in multiple investigated criterions. For the first time, excellent performance stability was demonstrated under various operational conditions and reusability over multiple extraction and regeneration cycles. Furthermore, to provide insights into the molecular mechanisms of extraction, computational quantum chemistry modeling was employed, which showed a strong agreement with the experimental results. The development of these new neoteric solvents for furfural recovery from biowaste offers a highly effective, sustainable, and eco-friendly alternative to traditional solvents, representing a significant breakthrough in the field of renewable bioenergy production and sustainable materials recovery.

Thermal evaluation of plant biomass from the phytostabilisation of soils contaminated by potentially toxic elements

The combination of phytoremediation of soils contaminated by potentially toxic elements with energy production by combustion of the generated biomass can be a sustainable land management option, combining the production of renewable bioenergy with soil restoration while minimising energy consumption and CO2 emission. In this work, plant biomass from phytoremediation of soils contaminated by potentially toxic elements was studied as solid biofuel for combustion by thermal analysis and biomass composition. Six plant species were grown in two soils with differing degrees of contamination: Brassica juncea, Cynara cardunculus, Atriplex halimus, Nicotiana glauca, Dittrichia viscosa, Retama sphaerocarpa and Salvia rosmarinus. The composition of the plant biomass was characterised chemically and thermogravimetric analyses were performed for the mass loss (TG), derivative curves of mass loss (DTG) and temperature difference (DTA) signal. The cellulose concentration correlated with the parameters of the thermal analysis in the low temperature range (150–350 °C), while lignin correlated with the thermal parameters of the second peak in the high temperature range. Salvia rosmarinus and R. sphaerocarpa showed the best combustion characteristics according to the thermal profile and mineral residue results. The accumulation of potentially toxic elements in B. juncea grown in heavily contaminated soil led to a higher amount of residue at 750 °C, with a global activation energy lower than the one obtained when this species was grown in a soil with lower contamination. Therefore, the most beneficial combination of soil phytoremediation and energy production (combustion) that can be suggested would depend on the level of soil contamination: in heavily contaminated soil, phytostabilisation using R. sphaerocarpa and S. rosmarinus; in slightly contaminated soil, B. juncea due to its high energy of activation, although the concentrations of potentially toxic elements in the residue must be controlled, as well as possible particulate matter emissions during combustion.

Transgenic ZmMYB167 Miscanthus sinensis with increased lignin to boost bioenergy generation for the bioeconomy

BackgroundPerennial C4 grasses from the genus Miscanthus are widely regarded as leading and promising dedicated bioenergy crops due to their high biomass accumulation on marginal land with low environmental impacts and maintenance requirements over its productive life. There is an urgent socio-political and environmental need to ramp up the production of alternative, affordable and green bioenergy sources and to re-direct the net zero carbon emissions trajectory. Hence, up-scaling of Miscanthus cultivation as a source of biomass for renewable energy could play an important role to strategically address sustainable development goals for a growing bio-based economy. Certain Miscanthus sinensis genotypes are particularly interesting for their biomass productivity across a wide range of locations. As the aromatic biomass component lignin exhibits a higher energy density than cell wall polysaccharides and is generally used as an indicator for heating or calorific value, genetic engineering could be a feasible strategy to develop M. sinensis biomass with increased lignin content and thus improving the energetic value of the biomass.ResultsFor this purpose, transgenic M. sinensis were generated by Agrobacterium-mediated transformation for expression of ZmMYB167, a MYB transcription factor known for regulating lignin biosynthesis in C3 and C4 grasses. Four independent transgenic ZmMYB167 Miscanthus lines were obtained. Agronomic traits such as plant height, tillering and above-ground dry weight biomass of the transgenic plants were not different to that of wild-type control plants. Total lignin content of the transgenic plants was ~ 15–24% higher compared with control plants. However, the structural carbohydrates, glucan and xylan, were decreased by ~ 2–7% and ~ 8–10%, respectively, in the transgenic plants. Moreover, expression of ZmMYB167 in transgenic plants did not alter lignin composition, phenolic compounds or enzymatic saccharification efficiency yields but importantly improved total energy levels in Miscanthus biomass, equivalent to 10% higher energy yield per hectare.ConclusionsThis study highlights ZmMYB167 as a suitable target for genetic lignin bioengineering interventions aimed at advancing and developing lignocellulosic biomass supply chains for sustainable production of renewable bioenergy.

Action of silicon on the activity of antioxidant enzymes and on physiological mechanisms mitigates water deficit in sugarcane and energy cane plants

Production of sugarcane and more recently of energy cane strengthen renewable bioenergy production capacity. However, droughts resulting from climate change have limited the production of these crops. One of the strategies to attenuate water deficit damage in these crops is the use of silicate, which contributes to plant physiology. This strategy is likely to increase water use efficiency, thus promoting crop sustainability. Notwithstanding, studies on this issue are still incipient. This study assesses whether Si applied via fertigation and foliar spraying in the seedling production phase and as a complement after seedling transplanting to the soil is efficient in attenuating water deficit in sugarcane and energy cane. The study further elucidates physiological and biochemical mechanisms involved in this process. For this, the authors conducted two experiments: one with sugarcane and the other with energy cane. Treatments were arranged in randomized blocks with 5 replications, in a 2 × 2 factorial scheme. Factors consisted of the absence (-Si) and presence of Si (+ Si) applied via fertigation and foliar spraying; and two water regimes: 70% (without water deficit) and 30% (severe water deficit) of the soil water retention capacity. Silicon was supplied during the formation phase of presprouted seedlings and during the transplanting of seedlings to pots filled with samples of Entisol (Quartzipsamment). In these pots, water regimes were induced from 7 to 160 days after transplanting. Severe water deficit reduced the water content and water potential of plants. This situation induced oxidative stress and impaired gas exchange and photosynthetic water use efficiency, reducing plant growth. Silicon supply via fertigation in association with foliar spraying in the seedling formation phase with complementation after transplanting was efficient in increasing Si accumulation in the plants. Silicon was effective in attenuating severe water deficit damage up to initial culm formation through mechanisms that maintain water and physiological balance by favoring the antioxidant defense system in sugarcane and energy cane plants.

Production of medium chain fatty acids through co-fermentation of food waste and sewage sludge without external electron donors

Effective biowaste treatment with renewable bioenergy production has become attractive. In this study, chain-elongation (CE) technology was applied to convert food waste (FW) and sewage sludge (SS) into high value-added medium chain fatty acids (MCFAs) by co-fermentation, which not only reduces environmental pollution but also recovers valuable resources. Five groups of batch co-fermentation experiments were performed using FW and SS at different ratios (FW/SS based on chemical oxygen demand, COD) without external electron donors (EDs) to investigate the effects of different FW/SS ratios on the production of MCFAs. The results showed that 100 % FW (FW/SS=1:0) achieved the highest n-caproate concentration (396.37 ± 35.98 mg COD/L), followed by 75 % FW (FW/SS=3:1) (384.42 ± 28.00 mg COD/L). Moreover, 75 % FW (FW/SS=3:1) had higher n-caproate specificity (11.22 ± 3.81 %) than 100 % FW (7.52 ± 0.68 %). Thus, a semi-continuous co-fermentation experiment at a FW/SS ratio of 3:1 was performed for 90 days to explore the feasibility of long-term MCFAs production without external EDs. The results showed that the maximum MCFAs concentration, specificity, and production rate were 10441.31 mg COD/L, 58.20 %, and 250.59 mg COD/L·d−1, respectively. Among the three MCFAs, n-caproate was the most abundant, accounting for 60.28 %. Microbial community analysis revealed that four lactate-producing bacteria (Bifidobacterium, Olsenella, Atopobium, and Proteiniphilum) co-occurred with Caproiciproducens, developing a CE system driven by lactate as the ED. This study provides new insights into the recovery of resources from FW and SS.

MOF-Derived Co3O4 Nanoparticles Catalyzing Hydrothermal Deoxygenation of Fatty Acids for Alkane Production

Designing economical and nonprecious catalysts with a catalytic performance as good as that of noble metals is of great importance in future renewable bioenergy production. In this study, the metal–organic framework (MOF) was applied as a precursor template to synthesize Co3O4 nanoparticles with a carbon matrix shell (denoted as M-Co3O4). To select the synthesized optimal catalyst, stearic acid was chosen as the model reactant. The effects of catalyst dosage, methanol dosage, water dosage, temperature, and reaction time on catalytic efficiency were examined. Under the designed condition, M-Co3O4 exhibited high catalytic performance and the catalyst showed higher conversion of stearic acid (98.7%) and selectivity toward C8–C18 alkanes (92.2%) in comparison with Pt/C (95.8% conversion and 93.2% selectivity toward C8–C18). Furthermore, a series of characterization techniques such as scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), nitrogen adsorption isotherms (Brunauer–Emmett–Teller (BET) method), and thermogravimetric analysis (TGA) was applied to investigate the physicochemical properties of the catalysts. Finally, we proposed that decarbonization (deCO) could be the presumably mechanistic pathway for the production of C8–C18 alkanes from the decomposition of stearic acid.

Estimation and Spatial Mapping of Residue Biomass following CTL Harvesting in Pinus radiata Plantations: An Application of Harvester Data Analytics

The utilization of forest harvest residues for renewable bioenergy production and bioproducts has increasingly become an integrated part of forestry that helps to meet the needs of climate change mitigation and a future carbon neutral economy. An essential element in the planning of any harvesting residue recovery operation is a reliable assessment of the quantity and quality of residue biomass and its composition over a harvest area. With the now widely adopted cut-to-length (CTL) at the stump harvesting system in Pinus radiata plantations in Australia, harvesting residues left on site are significantly larger in quantity and spatially more dispersed over a harvest area in comparison to the more traditional whole-tree harvesting. The conventional approach of assessing forest harvest residues through sample plots, transects, or small study blocks has provided site-specific estimates of residue biomass. However, these estimates cannot be readily extrapolated over the plantation landscape, which varies in silviculture, site, and stand conditions. To overcome this limitation, this study relied on harvester data analytics to obtain spatially explicit estimates of residue biomass using an example data set from harvested plantations of three stand types: unthinned (T0), thinned once (T1), and thinned twice (T2). Three methods of integrating biomass equations with harvester data were compared for residue biomass estimation: (1) applying individual tree biomass equations to harvested stems, (2) applying stand-level biomass equations to gridded harvester data, and (3) integrating estimates from the first approach with recorded and estimated waste volumes of harvested stems. The estimates of total residue biomass obtained using the three methods through harvester data analytics varied between 56.2 and 156.4 t/ha in green weight across the three stand types. These estimates were validated indirectly through ex situ sample plots and proved to be comparable to the quantities of residue biomass assessed using conventional sample plots, transects, or small blocks following CTL harvesting of rotation age P. radiata plantations elsewhere in Australia. Among the three methods, the third method made the most intensive use of the harvester data and provided the most realistic estimates of residue biomass. Spatial mapping of the estimated total and component residue biomass will assist the operational planning of residue recovery and site-specific nutrient management for the long-term sustainability of P. radiata plantations.

Palm Oil Decanter Cake Wastes as Alternative Nutrient Sources and Biomass Support Particles for Production of Fungal Whole-Cell Lipase and Application as Low-Cost Biocatalyst for Biodiesel Production

This is the first report on the possible use of decanter cake waste (DCW) from palm oil industry as alternative nutrient sources and biomass support particles for whole-cell lipase production under solid-state fermentation (SSF) by newly isolated fungal Aspergillus sp. MS15 and their application as a low-cost and environment-friendly biocatalyst for biodiesel production. The results found that DCW supplemented with 0.1% K2HPO4, 0.05% MgSO4·7H2O, 1% peptone and 2% urea and pH adjusted to 6.0 was optimal for whole-cell lipase production. The optimal moisture content and fermentation temperature was 60% and 37.5 °C, respectively. Environmentally friendly biodiesel production, through either esterification or transesterification using whole-cell lipase immobilized on DCW as a biocatalyst, was optimized. The optimal reaction temperature for both reactions was 37 °C. The whole-cell lipase effectively esterified oleic acid into >95% biodiesel yield through esterification under optimal water activity at 0.71 and an optimal methanol to oleic acid molar ratio of 2:1, and also effectively transesterified palm oil under optimal water activity at 0.81 and an optimal methanol to oil molar ratio of 3:1. The fuel properties of produced biodiesel are close to the international biodiesel standards. These results have shown the circular utilization of palm oil mill waste for the low-cost production of an effective biocatalyst, and may contribute greatly to the sustainability of renewable bioenergy production.

From simple and specific zymographic detections to the annotation of a fungus Daldinia caldariorum D263 that encodes a wide range of highly bioactive cellulolytic enzymes

BackgroundLignocellulolytic enzymes are essential for agricultural waste disposal and production of renewable bioenergy. Many commercialized cellulase mixtures have been developed, mostly from saprophytic or endophytic fungal species. The cost of complete cellulose digestion is considerable because a wide range of cellulolytic enzymes is needed. However, most fungi can only produce limited range of highly bioactive cellulolytic enzymes. We aimed to investigate a simple yet specific method for discovering unique enzymes so that fungal species producing a diverse group of cellulolytic enzymes can be identified.ResultsThe culture medium of an endophytic fungus, Daldinia caldariorum D263, contained a complete set of cellulolytic enzymes capable of effectively digesting cellulose residues into glucose. By taking advantage of the unique product inhibition property of β-glucosidases, we have established an improved zymography method that can easily distinguish β-glucosidase and exoglucanase activity. Our zymography method revealed that D263 can secrete a wide range of highly bioactive cellulases. Analyzing the assembled genome of D263, we found over 100 potential genes for cellulolytic enzymes that are distinct from those of the commercially used fungal species Trichoderma reesei and Aspergillus niger. We further identified several of these cellulolytic enzymes by mass spectrometry.ConclusionsThe genome of Daldinia caldariorum D263 has been sequenced and annotated taking advantage of a simple yet specific zymography method followed by mass spectrometry analysis, and it appears to encode and secrete a wide range of bioactive cellulolytic enzymes. The genome and cellulolytic enzyme secretion of this unique endophytic fungus should be of value for identifying active cellulolytic enzymes that can facilitate conversion of agricultural wastes to fermentable sugars for the industrial production of biofuels.

Improvement of Renewable Bioenergy Production in Microbial Fuel Cells with Saponin Supplementation

Microbial fuel cell (MFC) is one of renewable biofuel production technology that directly converts biomass to electricity. Cellulosic biomass is particularly attractive renewable resources for its low cost and abundance and neutral carbon balance. However, methanogenesis remains as a major factor limiting MFC performance. The current study reports that saponin addition at 0.05% w/v dose to anolyte in MFCs inhibited methanogenesis and improves power generation and cellulose fermentation. Mediator-less two chamber H-type MFCs were prepared using rumen fluid as anode inocula at 20% v/v of anolyte to convert finely ground pine tree (Avicel) at 2%, w/v to electricity. Saponin was added to the anode of MFC at 0.005% or 0.05% v/v dosage for treatment. MFC power and current across an external resistor were measured daily for 10d. On d10, collected gases from anode compartment were measured for total gas volume and analyzed for gas composition on gas chromatography. Supplementation of saponin to MFC at 0.005% did not have any effects on electricity generation or biogas production and composition. Saponin at 0.05% dose reduced 10% of methane production and increased 40% of CO2 production and 6.4% of total gas production for 10d MFC operation. Voltage across resistor prior to treatment addition (d0) was 164.75 ± 9.07 mV. In control group, voltage across resistor did not change (P = 0.9153) with time course and mean was 167.8 ± 8.20 mV ranged from 157 to 174.5 mV during 10d operation. In 0.05% Saponin group, voltage across resistor increased (P 0.0001) after d2 and mean was 187.3 ± 4.30 mV ranged between 161.5 and 204.0 mV and the 10d mean of voltage across resistor in 0.05% Saponin was greater (P 0.0001) than in control group. 0.05% Saponin also had greater voltage across resistor at d5 (P = 0.0030) and d6 (P = 0.0246) than control. End point potential increased (P 0.0001) in 0.05% Saponin after d2. 0.05% Saponin had greater (P 0.0001) in 0.05% Saponin. Power density increased (P 0.0001) after d2 in 0.05% Saponin. 0.05% Saponin MFCs had greater (P 0.0001) overall mean of 10d operation. The current study provides strong background for potential use of saponin and saponin containing natural resources for methanogenesis inhibitor and cellulolysis enhancer in MFC and also cellulolysis reactors.

A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.

A submerged duckweed mutant with abundant starch accumulation for bioethanol production

AbstractDuckweed is one kind of promising bioenergy plant with prominent advantages such as fast growth rate and high starch content. However, almost all previous studies focused on the natural duckweed germplasms. In this study, heavy‐ion irradiation was used to establish a mutant library of Lemna aequinoctialis 6002, and one mutant named submarine‐1 (sub‐1) was screened, which could accumulate more starch but with smaller granules. Unexpectedly, under proper external growth conditions such as poor nutritional status and insufficient growth space, sub‐1 mutant would sink underwater due to formation of dense tissue structure and large amount of fine starch particles with the extension of cultivation time. The starch content in the sinking sub‐1 increased to over 45% (dry weight) and was 12% higher than the floating sub‐1, highlighting that submergence can be considered as a spontaneous and efficient indicator for screening of high‐starch duckweed. Additionally, the saccharification efficiency of starch and ethanol yield had increased in sub‐1 mutant compared to the wild type. Based on the unique characteristics of sub‐1 mutant, a cultivation model of submerged duckweed in a simulated aquaculture pond was designed to get more starch‐rich biomass, enabling effective production of renewable bioenergy.

Estimation of Sugarcane Yield Using a Machine Learning Approach Based on UAV-LiDAR Data

Sugarcane is a multifunctional crop mainly used for sugar and renewable bioenergy production. Accurate and timely estimation of the sugarcane yield before harvest plays a particularly important role in the management of agroecosystems. The rapid development of remote sensing technologies, especially Light Detecting and Ranging (LiDAR), significantly enhances aboveground fresh weight (AFW) estimations. In our study, we evaluated the capability of LiDAR mounted on an Unmanned Aerial Vehicle (UAV) in estimating the sugarcane AFW in Fusui county, Chongzuo city of Guangxi province, China. We measured the height and the fresh weight of sugarcane plants in 105 sampling plots, and eight variables were extracted from the field-based measurements. Six regression algorithms were used to build the sugarcane AFW model: multiple linear regression (MLR), stepwise multiple regression (SMR), generalized linear model (GLM), generalized boosted model (GBM), kernel-based regularized least squares (KRLS), and random forest regression (RFR). The results demonstrate that RFR (R2 = 0.96, RMSE = 1.27 kg m−2) performs better than other models in terms of prediction accuracy. The final fitted sugarcane AFW distribution maps exhibited good agreement with the observed values (R2 = 0.97, RMSE = 1.33 kg m−2). Canopy cover, the distance to the road, and tillage methods all have an impact on sugarcane AFW. Our study provides guidance for calculating the optimum planting density, reducing the negative impact of human activities, and selecting suitable tillage methods in actual cultivation and production.

Long-term medium chain carboxylic acids production from liquor-making wastewater: Parameters optimization and toxicity mitigation

Medium chain carboxylic acids (MCCAs) production from waste biomass by an emerging open culture chain elongation (CE) technology is a sustainable and affordable approach for future production of renewable bioenergy. However, the toxicity of undissociated MCCAs limits MCCAs production and the stable operation of CE reactor. By utilizing expanded granular sludge bed (EGSB) as a CE reactor and Chinese liquor-making wastewater (CLMW) as feedstock, this study obtained currently highest MCCAs production rate (31.12 g COD/(L·d)) from real waste biomass with a MCCAs yield of 76.80%, and achieved a long-term and stable MCCAs production (485 days). The stable MCCAs production could be primarily attributed to the mitigation of toxic effects of undissociated MCCAs on CE bacteria due to the special structure of granule sludge. In addition, the high cell density and quick mass transfer efficiency of EGSB, the optimized operating parameters, and the efficiently shaped microbiome dominated by CE functional bacteria also contributed to the high MCCAs production. This work showed a potential for MCCAs production from CLMW and lay a foundation for scaling up MCCAs production by EGSB reactor.

Lignin Reactions and Structural Alternations under Typical Biomass Pretreatment Methods

The utilization of biomass in the production of renewable bioenergy and biomaterials has been a popular topic since the past decades as they are rich in carbohydrates. Most biomasses, such as wood, monocotyledons, and agriculture residues, need to be pretreated before the conversion of carbohydrates in order to break down the recalcitrant cell wall structure and increase the fiber accessibility. To date, a variety of pretreatment methods have been developed that vary from physical to chemical and biological methods. Pretreatment processes affect the cell wall physical structure as well as the chemical structure of the cell wall constituents. Comparing to the studies of the cellulose and hemicelluloses structural changes during pretreatment, such studies on lignin are relatively limited. On the other hand, in order to utilize the part of lignin from biorefinery processes, the understanding of the lignin structural changes during the refining process becomes important. In this study, typical pretreatment methods such as hydrothermal pretreatment, alkaline pretreatment, biodegradation, and oxidative pretreatment are introduced and their corresponding impacts on the lignin structures are reviewed.

Advantages a nd Problems o f t he Development o f Efficient Biofuel Production

The article discusses the advantages and problems of the development of efficient biofuel production. The experience of developed countries in the production of biofuels from agricultural raw materials. Defined economic and social aspects. There is a political side to the production of renewable bioenergy. The availability of verified energy sources gives the country a certain temporary "margin of energy durability" in the event of a sudden interruption in the supply of traditional fuels (oil, fuel oil, gas) and some opportunity for maneuvering energy resources within the state. The development of bioenergy based on the use of its own raw material base is economically feasible. This is not only biofuels and fertilizers, but also high-protein feed for livestock and poultry, as well as the possibility of energy supply of specific agricultural enterprises.

Engineering Brevibacterium flavum for the production of renewable bioenergy: C4-C5 advanced alcohols.

Biosynthesis of advanced biofuels by engineered non-natural microorganisms has been proposed to be the most promising approach for the replacement of dwindling fossil fuel resources. Brevibacterium flavum (Bf) is a model brevibacterium aerobe which lacks basic and applied research that could enable this species to produce biofuels. There are no reports regarding engineering this microorganism to produce advanced alcohols before. Here, for the first time, we developed the bacterium as a novel biosynthetic platform for advanced alcohols production via the mutagenesis and engineering to produce 2-ketoacids derived alcohols. In order to enhance the strain's capability of producing advanced alcohols, we preferentially improved intrinsic metabolism ability of the strain to obtain improved expression host (IEH) via generating mutagenesis libraries by whole cell mutagenesis (WCM). The IEH was determined via screening out the mutant strain with the highest production of branched-chain organic acids (BCOA) using high throughput screening method.. Subsequently, a novel vector system for Bf was established, and the corresponding biosynthetic pathway of directing carbon flux into the target advanced alcohols was recruited to make the bacterium possess the capability of producing advanced alcohols and further enhance the production using the IEH. Specifically, we generated bioengineered strains that were able to synthesize up to the highest 5362 and 4976 mg/L isobutanol, 1945 and 1747 mg/L 2-methyl-1-butanol (2 MB), and 785.34 and 781 mg/L 3-methyl-1-butanol (3 MB) from pure glucose and duckweed substrates, respectively. Our findings confirmed the feasibility and potential of using Bf as a novel biosynthetic platform to generate advanced biofuels with glucose and inexpensive renewable feedstock-duckweed as a fermentation substrate. Biotechnol. Bioeng. 2017;114: 1946-1958. © 2017 Wiley Periodicals, Inc.

Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis

The production of renewable bioenergy will be necessary to meet rising global fossil fuel demands. Members of the marine microalgae genus Nannochloropsis produce large quantities of oils (triacylglycerols; TAGs), and this genus is regarded as one of the most promising for biodiesel production. Recent genome sequencing and transcriptomic studies on Nannochloropsis have provided a foundation for understanding its oleaginous trait, but the mechanism underlying oil accumulation remains to be clarified. Here we report Nannochloropsis knock-out strains of four extraplastidic lysophosphatidic acid acyltransferases (LPAT1-LPAT4) that catalyze a major de novo biosynthetic step of TAGs and membrane lipids. We found that the four LPATs are differently involved in lipid metabolic flow in Nannochloropsis. Double knock-outs among the LPATs revealed the pivotal LPATs for TAG biosynthesis, and localization analysis indicated that the stramenopile-specific LPATs (LPAT3 and LPAT4) associated with TAG synthesis reside at the perimeter of lipid droplets. No homologous region has been found with other lipid droplet-associated proteins, however. Lipid droplets are an organelle found in nearly all organisms, and recently they were shown to play important roles in cellular metabolism and signaling. Our results provide direct evidence for the importance of the perimeter of lipid droplet in TAG synthesis in addition to its known role in maintaining TAG stability, and these findings suggest that the oleaginous trait of Nannochloropsis is enabled by the acquisition of LPATs at the perimeter of lipid droplets.

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.

Activation of lignocellulosic biomass for higher sugar yields using aqueous ionic liquid at low severity process conditions.

BackgroundConcerns around greenhouse gas emissions necessitate the development of sustainable processes for the production of chemicals, materials, and fuels from alternative renewable sources. The lignocellulosic plant cell walls are one of the most abundant sources of carbon for renewable bioenergy production. Certain ionic liquids (ILs) are very effective at disrupting the plant cell walls of lignocellulose, and generate a substrate that is effectively hydrolyzed into fermentable sugars. Conventional ILs are relatively expensive in terms of purchase price, and the most effective imidazolium-based ILs also require energy intensive processing conditions (>140 °C, 3 h) to release >90 % fermentable sugar yields after saccharification.ResultsWe have developed a highly effective pretreatment technology utilizing the relatively inexpensive IL comprised tetrabutylammonium [TBA]+ and hydroxide [OH]− ions that generate high glucose yields (~95 %) after pretreatment at very mild processing conditions (50 °C). The efficiency of [TBA][OH] pretreatment of lignocellulose was further studied by analyzing chemical composition, powder X-ray diffraction for cellulose structure, NMR and SEC for lignin dissolution/depolymerization, and glycome profiling for cell wall modifications. Glycome profiling experiments and computational results indicate that removal of the noncellulosic polysaccharides occurs due to the ionic mobility of [TBA][OH] and is the key factor in determining pretreatment efficiency. Process modeling and energy demand analysis suggests that this [TBA][OH] pretreatment could potentially reduce the energy required in the pretreatment unit operation by more than 75 %.ConclusionsBy leveraging the benefits of ILs that are effective at very mild processing conditions, such as [TBA][OH], lignocellulosic biomass can be pretreated at similar efficiency as top performing conventional ILs, such as 1-ethyl-3-methylimidazolium acetate [C2C1Im][OAc], but at much lower temperatures, and with less than half the IL normally required to be effective. [TBA][OH] IL is more reactive in terms of ionic mobility which extends removal of lignin and noncellulosic components of biomass at the lower temperature pretreatment. This approach to biomass pretreatment at lower temperatures could be transformative in the affordability and energy efficiency of lignocellulosic biorefineries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0561-7) contains supplementary material, which is available to authorized users.