Biofuels and value-added biochemicals derived from renewable biomass via biochemical conversion have attracted considerable attention to meet global sustainable energy and environmental goals. Isobutanol is a four-carbon alcohol with many advantages that make it attractive as a fossil-fuel alternative. Zymomonas mobilis is a highly efficient, anaerobic, ethanologenic bacterium making it a promising industrial platform for use in a biorefinery. In this study, the effect of isobutanol on Z. mobilis was investigated, and various isobutanol-producing recombinant strains were constructed. The results showed that the Z. mobilis parental strain was able to grow in the presence of isobutanol below 12 g/L while concentrations greater than 16 g/L inhibited cell growth. Integration of the heterologous gene encoding 2-ketoisovalerate decarboxylase such as kdcA from Lactococcus lactis is required for isobutanol production in Z. mobilis. Moreover, isobutanol production increased from nearly zero to 100–150 mg/L in recombinant strains containing the kdcA gene driven by the tetracycline-inducible promoter Ptet. In addition, we determined that overexpression of a heterologous als gene and two native genes (ilvC and ilvD) involved in valine metabolism in a recombinant Z. mobilis strain expressing kdcA can divert pyruvate from ethanol production to isobutanol biosynthesis. This engineering improved isobutanol production to above 1 g/L. Finally, recombinant strains containing both a synthetic operon, als-ilvC-ilvD, driven by Ptet and the kdcA gene driven by the constitutive strong promoter, Pgap, were determined to greatly enhance isobutanol production with a maximum titer about 4.0 g/L. Finally, isobutanol production was negatively affected by aeration with more isobutanol being produced in more poorly aerated flasks. This study demonstrated that overexpression of kdcA in combination with a synthetic heterologous operon, als-ilvC-ilvD, is crucial for diverting pyruvate from ethanol production for enhanced isobutanol biosynthesis. Moreover, this study also provides a strategy for harnessing the valine metabolic pathway for future production of other pyruvate-derived biochemicals in Z. mobilis.

The growth of the cellulosic ethanol industry is currently impeded by high production costs. One possible solution is to improve the performance of fermentation itself, which has great potential to improve the economics of the entire production process. Here, we demonstrated significantly improved productivity through application of an advanced fermentation approach, named self-cycling fermentation (SCF), for cellulosic ethanol production. The flow rate of outlet gas from the fermenter was used as a real-time monitoring parameter to drive the cycling of the ethanol fermentation process. Then, long-term operation of SCF under anaerobic conditions was improved by the addition of ergosterol and fatty acids, which stabilized operation and reduced fermentation time. Finally, an automated SCF system was successfully operated for 21 cycles, with robust behavior and stable ethanol production. SCF maintained similar ethanol titers to batch operation while significantly reducing fermentation and down times. This led to significant improvements in ethanol volumetric productivity (the amount of ethanol produced by a cycle per working volume per cycle time)—ranging from 37.5 to 75.3%, depending on the cycle number, and in annual ethanol productivity (the amount of ethanol that can be produced each year at large scale)—reaching 75.8 ± 2.9%. Improved flocculation, with potential advantages for biomass removal and reduction in downstream costs, was also observed. Our successful demonstration of SCF could help reduce production costs for the cellulosic ethanol industry through improved productivity and automated operation.

Lignocellulosic biomass has been commonly regarded as a potential feedstock for the production of biofuels and biochemicals. High sugar yields and the complete bioconversion of all the lignocellulosic sugars into valuable products are attractive for the utilization of lignocelluloses. It is essential to pretreat and hydrolyze lignocelluloses at high solids loadings during industrial processes, which is more economical and environmentally friendly as capital cost, energy consumption, and water usage can be reduced. However, oligosaccharides are inevitably released during the high solids loading enzymatic hydrolysis and they are more recalcitrant than monosaccharides for microorganisms. A fed-batch enzymatic hydrolysis of corn stover pretreated by the sodium hydroxide–methanol solution (SMs) at high solids loading was demonstrated to reach the high concentrations and yields of fermentable sugars. Glucose, xylose, cello-oligosaccharides, and xylo-oligosaccharides achieved 146.7 g/L, 58.7 g/L, 15.6 g/L, and 24.7 g/L, respectively, when the fed-batch hydrolysis was started at 12% (w/v) solids loading, and 7% fresh substrate and a standardized blend of cellulase, β-glucosidase, and hemicellulase were fed consecutively at 3, 6, 24, and 48 h to achieve a final solids loading of 40% (w/v). The total conversion of glucan and xylan reached 89.5% and 88.5%, respectively, when the oligosaccharides were taken into account. Then, a fed-batch culture on the hydrolysates was investigated for lipid production by Cutaneotrichosporon oleaginosum. Biomass, lipid content, and lipid yield were 50.7 g/L, 61.7%, and 0.18 g/g, respectively. The overall consumptions of cello-oligosaccharides and xylo-oligosaccharides reached 74.1% and 68.2%, respectively. High sugars concentrations and yields were achieved when the enzyme blend was supplemented simultaneously with the substrate at each time point of feeding during the fed-batch enzymatic hydrolysis. Oligosaccharides were co-utilized with monosaccharides during the fed-batch culture of C. oleaginosum. These results provide a promising strategy to hydrolyze alkaline organosolv-pretreated corn stover into fermentable sugars with high concentrations and yields for microbial lipid production.

Lignocellulosic biorefinery offers economical and sustainable production of fuels and chemicals. Saccharomyces cerevisiae, a promising industrial host for biorefinery, has been intensively developed to expand its product profile. However, the sequential and slow conversion of xylose into target products remains one of the main challenges for realizing efficient industrial lignocellulosic biorefinery. In this study, we developed a powerful mixed-sugar co-fermenting strain of S. cerevisiae, XUSEA, with improved xylose conversion capacity during simultaneous glucose/xylose co-fermentation. To reinforce xylose catabolism, the overexpression target in the pentose phosphate pathway was selected using a DNA assembler method and overexpressed increasing xylose consumption and ethanol production by twofold. The performance of the newly engineered strain with improved xylose catabolism was further boosted by elevating fermentation temperature and thus significantly reduced the co-fermentation time by half. Through combined efforts of reinforcing the pathway of xylose catabolism and elevating the fermentation temperature, XUSEA achieved simultaneous co-fermentation of lignocellulosic hydrolysates, composed of 39.6 g L−1 glucose and 23.1 g L−1 xylose, within 24 h producing 30.1 g L−1 ethanol with a yield of 0.48 g g−1. Owing to its superior co-fermentation performance and ability for further engineering, XUSEA has potential as a platform in a lignocellulosic biorefinery toward realizing a more economical and sustainable process for large-scale bioethanol production.

As a leading biomass feedstock, poplar plants provide enormous lignocellulose resource convertible for biofuels and bio-chemicals. However, lignocellulose recalcitrance particularly in wood plants, basically causes a costly bioethanol production unacceptable for commercial marketing with potential secondary pollution to the environment. Therefore, it becomes important to reduce lignocellulose recalcitrance by genetic modification of plant cell walls, and meanwhile to establish advanced biomass process technology in woody plants. Brassinosteroids, plant-specific steroid hormones, are considered to participate in plant growth and development for biomass production, but little has been reported about brassinosteroids roles in plant cell wall assembly and modification. In this study, we generated transgenic poplar plant that overexpressed DEETIOLATED2 gene for brassinosteroids overproduction. We then detected cell wall feature alteration and examined biomass enzymatic saccharification for bioethanol production under various chemical pretreatments. Compared with wild type, the PtoDET2 overexpressed transgenic plants contained much higher brassinosteroids levels. The transgenic poplar also exhibited significantly enhanced plant growth rate and biomass yield by increasing xylem development and cell wall polymer deposition. Meanwhile, the transgenic plants showed significantly improved lignocellulose features such as reduced cellulose crystalline index and degree of polymerization values and decreased hemicellulose xylose/arabinose ratio for raised biomass porosity and accessibility, which led to integrated enhancement on biomass enzymatic saccharification and bioethanol yield under various chemical pretreatments. In contrast, the CRISPR/Cas9-generated mutation of PtoDET2 showed significantly lower brassinosteroids level for reduced biomass saccharification and bioethanol yield, compared to the wild type. Notably, the optimal green-like pretreatment could even achieve the highest bioethanol yield by effective lignin extraction in the transgenic plant. Hence, this study proposed a mechanistic model elucidating how brassinosteroid regulates cell wall modification for reduced lignocellulose recalcitrance and increased biomass porosity and accessibility for high bioethanol production. This study has demonstrated a powerful strategy to enhance cellulosic bioethanol production by regulating brassinosteroid biosynthesis for reducing lignocellulose recalcitrance in the transgenic poplar plants. It has also provided a green-like process for biomass pretreatment and enzymatic saccharification in poplar and beyond.

Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WTintact), the catalytically deficient mutant Cel7A E212Q (E212Qintact) and their proteolytically isolated catalytic domains (CD) (WTcore and E212Qcore, respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy. Binding of WTintact and E212Qintact on the recalcitrant algal cellulose (AC) showed two bound populations: ~ 85% bound with shorter residence times of < 15 s while ~ 15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WTcore and E212Qcore, exhibited three binding populations on AC: ~ 75% bound with short residence times of ~ 15 s (similar to the intact enzymes), ~ 20% bound for < 100 s and ~ 5% that were effectively immobilized. Cel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.

It has been argued that the US biofuel policy is responsible for the land use changes in Malaysia and Indonesia (M&I). In this paper, following a short literature review that highlights the relevant topics and issues, we develop analytical and numerical analyses to evaluate the extent to which production of biofuels in the US alters land use in M&I. The analytical analyses make it clear that market-mediated responses may generate some land use change in M&I due to biofuel production in the US. These analyses highlight the role of substitution among vegetable oils in linking these economies in markets for vegetable oils. To numerically quantify these effects, we modified and used a well-known Computable General Equilibrium model (CGE), GTAP-BIO. We conducted some sensitivity tests as well. According to the simulation results obtained from two base case scenarios for corn ethanol and soy biodiesel, we find that producing 15 BGs of corn ethanol and 2 BGs gallons of soy biodiesel together could potentially increase area of cropland in M&I by 59.6 thousand hectares. That is less than 0.5% of the cropland expansion in M&I for the time period of 2000–2016, when biofuel production increased in the US. The original GTAP-BIO model parameters including the regional substitution rates among vegetable oils were used for the base case scenarios. The estimated induced land use change (ILUC) emissions values for corn ethanol and soy biodiesel are about 12.3 g CO2e MJ−1, 17.5 g CO2e MJ−1 for the base case scenarios. The share of M&I in the estimated ILUC emissions value for corn ethanol is 10.9%. The corresponding figure for soy biodiesel is much higher, 78%. The estimated ILUC emissions value for soy biodiesel is sensitive with respect to the changes in the regional rates of substitution elasticity among vegetable oils. That is not the case for corn ethanol. When we replaced the original substitution elasticities of the base case, which are very large (i.e., 5 or 10) for many regions, with a small and uniform rate of substitution (i.e., 0.5) across the world, the ILUC emissions value for soy biodiesel drops from 17.5 g CO2e MJ−1 to 10.16 g CO2e MJ−1. When we applied larger substitution elasticities among vegetable oils, the estimated ILUC emissions value for soy biodiesel converged towards the base case results. This suggests that, other factors being equal, the base case substitution elasticities provide the largest possible ILUC emissions value for soy biodiesel. Finally, our analyses clearly indicate that those analyses that limit their modeling framework to only palm and soy oil and ignore other types of vegetable oils and fats provide misleading information and exaggerate about the land use implications of the US biofuels for M&I. (1) Production of biofuels in the US generates some land use effects in M&I due to market-mediated responses, in particular through the links between markets for vegetable oils. These effects are minor compared to the magnitude of land use change in M&I. However, because of the high carbon intensity of the peatland the emissions fraction of M&I is larger, in particular for soy biodiesel. (2) The GTAP-BIO model implemented a set of regional substitution elasticities among vegetable oils that, other factors being equal, provides the largest possible ILUC emissions value for soy biodiesel. (3) With a larger substitution elasticity among all types of vegetable oils and animal fats in the US, less land use changes occur in M&I. That is due to the fact that a larger substitution elasticity among vegetable oils in the US, diverts a larger portion of the additional demand for soy oil to non-palm vegetable oils and animal fats that are produced either in the US or regions other than M&I. (4) Those analyses that limit their modeling framework to only palm and soy oils and ignore other types of vegetable oils and fats provide misleading information and exaggerate about the land use implications of the US biofuels for M&I.

Omega-3 fatty acids have various health benefits in combating against neurological problems, cancers, cardiac problems and hypertriglyceridemia. The main dietary omega-3 fatty acids are obtained from marine fish. Due to the pollution of marine environment, recently microalgae are considered as the promising source for the omega-3 fatty acid production. However, the demand and high production cost associated with microalgal biomass make it necessary to implement novel strategies in improving the biomass and omega-3 fatty acids from microalgae. Four plant hormones zeatin, indole acetic acid (IAA), gibberellic acid (GBA) and abscisic acid (ABA) were investigated for their effect on the production of biomass and lipid in isolated Chlorella sp. The cells showed an increase of the biomass and lipid content after treatments with the plant hormones where the highest stimulatory effect was observed in ABA-treated cells. On the other hand, IAA showed the highest stimulatory effect on the omega-3 fatty acids content, eicosapentaenoic acid (EPA) (23.25%) and docosahexaenoic acid (DHA) (26.06%). On the other hand, cells treated with ABA had highest lipid content suitable for the biodiesel applications. The determination of ROS markers, antioxidant enzymes, and fatty acid biosynthesis genes after plant hormones treatment helped elucidate the mechanism underlying the improvement in biomass, lipid content and omega-3 fatty acids. All four plant hormones upregulated the fatty acid biosynthesis genes, whereas IAA particularly increased omega-3-fatty acids as a result of the upregulation of omega-3 fatty acid desaturase. The contents of omega-3 fatty acids, the clinically important compounds, were considerably improved in IAA-treated cells. The highest lipid content obtained from ABA-treated biomass can be used for biodiesel application according to its biodiesel properties. The EPA and DHA enriched ethyl esters are an approved form of omega-3 fatty acids by US Food and Drug Administration (FDA) which can be utilized as the therapeutic treatment for the severe hypertriglyceridemia.

As a renewable carbon source, biomass energy not only helps in resolving the management problems of lignocellulosic wastes, but also helps to alleviate the global climate change by controlling environmental pollution raised by their generation on a large scale. However, the bottleneck problem of extensive production of biofuels lies in the filamentous crystal structure of cellulose and the embedded connection with lignin in biomass that leads to poor accessibility, weak degradation and digestion by microorganisms. Some pretreatment methods have shown significant improvement of methane yield and production rate, but the promotion mechanism has not been thoroughly studied. Revealing the temporal and spatial effects of pretreatment on lignocellulose will greatly help deepen our understanding of the optimization mechanism of pretreatment, and promote efficient utilization of lignocellulosic biomass. Here, we propose an approach for qualitative, quantitative, and location analysis of subcellular lignocellulosic changes induced by alkali treatment based on label-free Raman microspectroscopy combined with chemometrics. Firstly, the variations of rice straw induced by alkali treatment were characterized by the Raman spectra, and the Raman fingerprint characteristics for classification of rice straw were captured. Then, a label-free Raman chemical imaging strategy was executed to obtain subcellular distribution of the lignocellulose, in the strategy a serious interference of plant tissues’ fluorescence background was effectively removed. Finally, the effects of alkali pretreatment on the subcellular spatial distribution of lignocellulose in different types of cells were discovered. The results demonstrated the mechanism of alkali treatment that promotes methane production in rice straw through anaerobic digestion by means of a systemic study of the evidence from the macroscopic measurement and Raman microscopic quantitative and localization two-angle views. Raman chemical imaging combined with chemometrics could nondestructively realize qualitative, quantitative, and location analysis of the lignocellulose of rice straw at a subcellular level in a label-free way, which was beneficial to optimize pretreatment for the improvement of biomass conversion efficiency and promote extensive utilization of biofuel.

Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts. We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal. This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.

Filamentous fungi have the ability to efficiently decompose plant biomass, and thus are widely used in the biofuel and bioprocess industries. In process, ambient pH has been reported to strongly affect the performance of the applied functional filamentous fungi. In this study, Trichoderma guizhouense NJAU4742 was investigated under the fermentation of rice straw at different initial pH values for a detailed study. The results showed that NJAU4742 strain could tolerate ambient pH values ranging from 3.0 to 9.0, but had significantly higher growth speed and extracellular enzyme activities under acidic conditions. At low ambient pH (< 4), NJAU4742 strain achieved rapid degradation of rice straw by elevating the ambient pH to an optimal range through environmental alkalinization. Further proteomic analysis identified a total of 1139 intracellular and extracellular proteins during the solid-state fermentation processes, including the quantified 190 carbohydrate-active enzymes (CAZymes) responsible for rice straw degradation, such as 19 cellulases, 47 hemicellulases and 11 chitinases. Meanwhile, the analysis results clearly showed that the secreted lignocellulases had a synergistic trend in distribution according to the ambient pH, and thus led to a pH-dependent classification of lignocellulases in T. guizhouense NJAU4742. Most functional lignocellulases were found to be differently regulated by the ambient pH in T. guizhouense NJAU4742, which had the ability of speeding up biomass degradation by elevating the ambient pH through environmental alkalinization. These findings contribute to the theoretical basis for the biodegradation of plant biomass by filamentous fungi in the biofuel and bioprocess industries.

Volatile fatty acids (VFAs) can be effective and promising alternate carbon sources for microbial lipid production by a few oleaginous yeasts. However, the severe inhibitory effect of high-content (> 10 g/L) VFAs on these yeasts has impeded the production of high lipid yields and their large-scale application. Slightly acidic conditions have been commonly adopted because they have been considered favorable to oleaginous yeast cultivation. However, the acidic pH environment further aggravates this inhibition because VFAs appear largely in an undissociated form under this condition. Alkaline conditions likely alleviate the severe inhibition of high-content VFAs by significantly increasing the dissociation degree of VFAs. This hypothesis should be verified through a systematic research. The combined effects of high acetic acid concentrations and alkaline conditions on VFA utilization, cell growth, and lipid accumulation of Yarrowia lipolytica were systematically investigated through batch cultures of Y. lipolytica by using high concentrations (30–110 g/L) of acetic acid as a carbon source at an initial pH ranging from 6 to 10. An initial pH of 8 was determined as optimal. The highest biomass and lipid production (37.14 and 10.11 g/L) were obtained with 70 g/L acetic acid, whereas cultures with > 70 g/L acetic acid had decreased biomass and lipid yield due to excessive anion accumulation. Feasibilities on high-content propionic acid, butyric acid, and mixed VFAs were compared and evaluated. Results indicated that YX/S and YL/S of cultures on butyric acid (0.570, 0.144) were comparable with those on acetic acid (0.578, 0.160) under alkaline conditions. The performance on propionic acid was much inferior to that on other acids. Mixed VFAs were more beneficial to fast adaptation and lipid production than single types of VFA. Furthermore, cultures on food waste (FW) and fruit and vegetable waste (FVW) fermentate were carried out and lipid production was effectively improved under this alkaline condition. The highest biomass and lipid production on FW fermentate reached 14.65 g/L (YX/S: 0.414) and 3.20 g/L (YL/S: 0.091) with a lipid content of 21.86%, respectively. By comparison, the highest biomass and lipid production on FVW fermentate were 11.84 g/L (YX/S: 0.534) and 3.08 g/L (YL/S: 0.139), respectively, with a lipid content of 26.02%. This study assumed and verified that alkaline conditions (optimal pH 8) could effectively alleviate the lethal effect of high-content VFA on Y. lipolytica and significantly improve biomass and lipid production. These results could provide a new cultivation strategy to achieve simple utilizations of high-content VFAs and increase lipid production. Feasibilities on FW and FVW-derived VFAs were evaluated, and meaningful information was provided for practical applications.

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.

Lignocellulosic biomass, which is composed of cellulose, hemicellulose and lignin, represents the most abundant renewable carbon source with significant potential for the production of sustainable chemicals and fuels. Current biorefineries focus on cellulose and hemicellulose valorization, whereas lignin is treated as a waste product and is burned to supply energy to the biorefineries. The depolymerization of lignin into well-defined mono-aromatic chemicals suitable for downstream processing is recognized increasingly as an important starting point for lignin valorization. In this study, conversion of all three components of Eucalyptus grandis into the corresponding monomeric chemicals was investigated using solid and acidic catalyst in sequence. Lignin was depolymerized into well-defined monomeric phenols in the first step using a Pd/C catalyst. The maximum phenolic monomers yield of 49.8 wt% was achieved at 240 °C for 4 h under 30 atm H2. In the monomers, 4-propanol guaiacol (12.9 wt%) and 4-propanol syringol (31.9 wt%) were identified as the two major phenolic products with 90% selectivity. High retention of cellulose and hemicellulose pulp was also obtained, which was treated with FeCl3 catalyst to attain 5-hydroxymethylfurfural, levulinic acid and furfural simultaneously. The optimal reaction condition for the co-conversion of hemicellulose and cellulose was established as 190 °C and 100 min, from which furfural and levulinic acid were obtained in 55.9% and 73.6% yields, respectively. Ultimately, 54% of Eucalyptus sawdust can be converted into well-defined chemicals under such an integrated biorefinery method. A two-step process (reductive catalytic fractionation followed by FeCl3 catalysis) allows the fractionation of all the three biopolymers (cellulose, hemicellulose, and lignin) in Eucalyptus biomass, which provides a promising strategy to make high-value chemicals from sustainable biomass.

The critical issue in the competitiveness between bioengineering and chemical engineering is the products titer and the volume productivity. The most direct and effective approach usually employs high-density biocatalyst, while the weakened mass transfer and evoked foam problem accompany ultrahigh-density biocatalyst loading and substrate/product titer. In high-density obligate aerobic bioconversion, oxygen as electron acceptor is a speed-limiting step in bioprocesses, but sufficient oxygen supply will lead to the foaming which results in a significant reduction in oxygen utilization and the use of additional defoamers. In this study, we designed a novel sealed-oxygen supply (SOS) biotechnology to resolve the formidable barrier of oxygen transferring rate (OTR), for bio-based fuels and chemical production process. Based on systemic analysis of whole-cell catalysis in Gluconobacter oxydans, a novel sealed-oxygen supply technology was smartly designed and experimentally performed for biocatalytic oxidation of alcohols, sugars and so on. By a simple operation skill of automatic online supply of oxygen in a sealed stirring tank bioreactor of SOS, OTR barrier and foaming problem was resolved with great ease. We finally obtained ultrahigh-titer products of xylonic acid (XA), 3-hydroxypropionic acid (3-HPA), and erythrulose at 588.4 g/L, 69.4 g/L, and 364.7 g/L, respectively. Moreover, the volume productivity of three chemical products was improved by 150–250% compared with normal biotechnology. This SOS technology provides a promising approach to promote bioengineering competitiveness and advantages over chemical engineering. SOS technology was demonstrated as an economic and universally applicable approach to bio-based fuels and chemicals production by whole-cell catalysis. The novel technology greatly promotes the competitiveness of bioengineering for chemical engineering, and provides a promising platform for the green and environmental use of biofuels.

The bioconversion of lignocellulose to fermentable C5/C6-saccharides is composed of pretreatment and enzymatic hydrolysis. Lignin, as one of the main components, resists lignocellulose to be bio-digested. Alkali and organosolv treatments were reported to be able to delignify feedstocks and loose lignocellulose structure. In addition, the use of additives was an alternative way to block lignin and reduce the binding of cellulases to lignin during hydrolysis. However, the relatively high cost of these additives limits their commercial application. This study explored the feasibility of using elephant grass (Pennisetum purpureum) and reed straw (Phragmites australis), both of which are important fibrous plants with high biomass, no-occupation of cultivated land, and soil phytoremediation, as feedstocks for bio-saccharification. Compared with typical agricultural residues, elephant grass and reed straw contained high contents of cellulose and hemicellulose. However, lignin droplets on the surface of elephant grass and the high lignin content in reed straw limited their hydrolysis performances. High hydrolysis yield was obtained for reed straw after organosolv and alkali pretreatments via increasing cellulose content and removing lignin. However, the hydrolysis of elephant grass was only enhanced by organosolv pretreatment. Further study showed that the addition of bovine serum albumin (BSA) or thioredoxin with His- and S-Tags (Trx-His-S) improved the hydrolysis of alkali-pretreated elephant grass. In particular, Trx-His-S was first used as an additive in lignocellulose saccharification. Its structural and catalytic properties were supposed to be beneficial for enzymatic hydrolysis. Elephant grass and reed straw could be used as feedstocks for bioconversion. Organosolv and alkali pretreatments improved their enzymatic sugar production; however, the increase in hydrolysis yield of pretreated elephant grass was not as effective as that of reed straw. During the hydrolysis of alkali-pretreated elephant grass, Trx-His-S performed well as additive, and its structural and catalytic capability was beneficial for enzymatic hydrolysis.

Programmed cell death (PCD) induced by acetic acid, the main by-product released during cellulosic hydrolysis, cast a cloud over lignocellulosic biofuel fermented by Saccharomyces cerevisiae and became a burning problem. Atg22p, an ignored integral membrane protein located in vacuole belongs to autophagy-related genes family; prior study recently reported that it is required for autophagic degradation and efflux of amino acids from vacuole to cytoplasm. It may alleviate the intracellular starvation of nutrition caused by Ac and increase cell tolerance. Therefore, we investigate the role of atg22 in cell death process induced by Ac in which attempt is made to discover new perspectives for better understanding of the mechanisms behind tolerance and more robust industrial strain construction. In this study, we compared cell growth, physiological changes in the absence and presence of Atg22p under Ac exposure conditions. It is observed that disruption and overexpression of Atg22p delays and enhances acetic acid-induced PCD, respectively. The deletion of Atg22p in S. cerevisiae maintains cell wall integrity, and protects cytomembrane integrity, fluidity and permeability upon Ac stress by changing cytomembrane phospholipids, sterols and fatty acids. More interestingly, atg22 deletion increases intracellular amino acids to aid yeast cells for tackling amino acid starvation and intracellular acidification. Further, atg22 deletion upregulates series of stress response genes expression such as heat shock protein family, cell wall integrity and autophagy. The findings show that Atg22p possessed the new function related to cell resistance to Ac. This may help us have a deeper understanding of PCD induced by Ac and provide a new strategy to improve Ac resistance in designing industrial yeast strains for bioethanol production during lignocellulosic biofuel fermentation.

Enzymatic hydrolysis continues to have a significant projected production cost for the biological conversion of biomass to fuels and chemicals, motivating research into improved enzyme and reactor technologies in order to reduce enzyme usage and equipment costs. However, technology development is stymied by a lack of accurate and computationally accessible enzymatic-hydrolysis reaction models. Enzymatic deconstruction of cellulosic materials is an exceedingly complex physico-chemical process. Models which elucidate specific mechanisms of deconstruction are often too computationally intensive to be accessible in process or multi-physics simulations, and empirical models are often too inflexible to be effectively applied outside of their batch contexts. In this paper, we employ a phenomenological modeling approach to represent rate slowdown due to substrate structure (implemented as two substrate phases) and feedback inhibition, and apply the model to a continuous reactor system. A phenomenological model was developed in order to predict glucose and solids concentrations in batch and continuous enzymatic-hydrolysis reactors from which liquor is continuously removed by ultrafiltration. A series of batch experiments were performed, varying initial conditions (solids, enzyme, and sugar concentrations), and best-fit model parameters were determined using constrained nonlinear least-squares methods. The model achieved a good fit for overall sugar yield and insoluble solids concentration, as well as for the reduced rate of sugar production over time. Additionally, without refitting model coefficients, good quantitative agreement was observed between results from continuous enzymatic-hydrolysis experiments and model predictions. Finally, the sensitivity of the model to its parameters is explored and discussed. Although the phenomena represented by the model correspond to behaviors that emerge from clusters of mechanisms, and hence a set of model coefficients are unique to the substrate and the enzyme system, the model is efficient to solve and may be applied to novel reactor schema and implemented in computational fluid dynamics (CFD) simulations. Hence, this modeling approach finds the right balance between model complexity and computational efficiency. These capabilities have broad application to reactor design, scale-up, and process optimization.

Pichia pastoris is becoming a promising chassis cell for metabolic engineering and synthetic biology after its whole genome and transcriptome sequenced. However, the current systems for multigene co-expression in P. pastoris are not efficient. The internal ribosome entry site (IRES) has an ability to recruit the ribosome to initiate protein synthesis by cap-independent translation manner. This study seeks to screen IRES sequences that are functional in P. pastoris, which will allow P. pastoris to express multiple proteins in a single mRNA and increase its efficacy as a platform for metabolic engineering and synthetic biology. In order to efficiently screen the IRES sequences, we first set out to create a screening system using LacZ gene. Due to the cryptic transcription of the LacZ gene, we established the α-complementation system of β-galactosidase in P. pastoris with the optimum length of the α-complementing peptide at ~ 92 amino acids. The optimal α-complementing peptide was then used as the second reporter to screen IRESes in the engineered GS115 expressing the corresponding ω-peptide. A total of 34 reported IRESes were screened. After ruling out all false positive or negative IRESes, only seven IRESes were functional in P. pastoris, which were from TEV, PVY, RhPV, TRV, KSHV, crTMV viruses and the 5′-UTR of the YAP1 gene of S. cerevisiae. We showed here that α-complementation also works in P. pastoris and it can be used in a variety of in vivo studies. The functional IRESes screened in this study can be used to introduce multiple genes into P. pastoris via a prokaryotic-like polycistronic manner, which provided new efficient tools for metabolic engineering and synthetic biology researches in P. pastoris.

Microalgae hold great promises as sustainable cellular factories for the production of alternative fuels, feeds, and biopharmaceuticals for human health. While the biorefinery approach for fuels along with the coproduction of high-value compounds with industrial, therapeutic, or nutraceutical applications have the potential to make algal biofuels more economically viable, a number of challenges continue to hamper algal production systems at all levels. One such hurdle includes the metabolic trade-off often observed between the increased yields of desired products, such as triacylglycerols (TAG), and the growth of an organism. Initial genetic engineering strategies to improve lipid productivity in microalgae, which focused on overproducing the enzymes involved in fatty acid and TAG biosynthesis or inactivating competing carbon (C) metabolism, have seen some successes albeit at the cost of often greatly reduced biomass. Emergent approaches that aim at modifying the dynamics of entire metabolic pathways by engineering of pertinent transcription factors or signaling networks appear to have successfully achieved a balance between growth and neutral lipid accumulation. However, the biological knowledge of key signaling networks and molecular components linking these two processes is still incomplete in photosynthetic eukaryotes, making it difficult to optimize metabolic engineering strategies for microalgae. Here, we focus on nitrogen (N) starvation of the model green microalga, Chlamydomonas reinhardtii, to present the current understanding of the nutrient-dependent switch between proliferation and quiescence, and the drastic reprogramming of metabolism that results in the storage of C compounds following N starvation. We discuss the potential components mediating the transcriptional repression of cell cycle genes and the establishment of quiescence in Chlamydomonas, and highlight the importance of signaling pathways such as those governed by the target of rapamycin (TOR) and sucrose nonfermenting-related (SnRK) kinases in the coordination of metabolic status with cellular growth. A better understanding of how the cell division cycle is regulated in response to nutrient scarcity and of the signaling pathways linking cellular growth to energy and lipid homeostasis, is essential to improve the prospects of biofuels and biomass production in microalgae.

Thermophilic filamentous fungus Myceliophthora thermophila has great capacity for biomass degradation and is an attractive system for direct production of enzymes and chemicals from plant biomass. Its industrial importance inspired us to develop genome editing tools to speed up the genetic engineering of this fungus. First-generation CRISPR–Cas9 technology was developed in 2017 and, since then, some progress has been made in thermophilic fungi genetic engineering, but a number of limitations remain. They include the need for complex independent expression cassettes for targeting multiplex genomic loci and the limited number of available selectable marker genes. In this study, we developed an Acidaminococcus sp. Cas12a-based CRISPR system for efficient multiplex genome editing, using a single-array approach in M. thermophila. These CRISPR–Cas12a cassettes worked well for simultaneous multiple gene deletions/insertions. We also developed a new simple approach for marker recycling that relied on the novel cleavage activity of the CRISPR–Cas12a system to make DNA breaks in selected markers. We demonstrated its performance by targeting nine genes involved in the cellulase production pathway in M. thermophila via three transformation rounds, using two selectable markers neo and bar. We obtained the nonuple mutant M9 in which protein productivity and lignocellulase activity were 9.0- and 18.5-fold higher than in the wild type. We conducted a parallel investigation using our transient CRISPR–Cas9 system and found the two technologies were complementary. Together we called them CRISPR–Cas-assisted marker recycling technology (Camr technology). Our study described new approaches (Camr technology) that allow easy and efficient marker recycling and iterative stacking of traits in the same thermophilic fungus strain either, using the newly established CRISPR–Cas12a system or the established CRISPR–Cas9 system. This Camr technology will be a versatile and efficient tool for engineering, theoretically, an unlimited number of genes in fungi. We expect this advance to accelerate biotechnology-oriented engineering processes in fungi.

The implementation of biorefineries based on lignocellulosic materials as an alternative to fossil-based refineries calls for efficient methods for fractionation and recovery of the products. The focus for the biorefinery concept for utilisation of biomass has shifted, from design of more or less energy-driven biorefineries, to much more versatile facilities where chemicals and energy carriers can be produced. The sugar-based biorefinery platform requires pretreatment of lignocellulosic materials, which can be very recalcitrant, to improve further processing through enzymatic hydrolysis, and for other downstream unit operations. This review summarises the development in the field of pretreatment (and to some extent, of fractionation) of various lignocellulosic materials. The number of publications indicates that biomass pretreatment plays a very important role for the biorefinery concept to be realised in full scale. The traditional pretreatment methods, for example, steam pretreatment (explosion), organosolv and hydrothermal treatment are covered in the review. In addition, the rapidly increasing interest for chemical treatment employing ionic liquids and deep-eutectic solvents are discussed and reviewed. It can be concluded that the huge variation of lignocellulosic materials makes it difficult to find a general process design for a biorefinery. Therefore, it is difficult to define “the best pretreatment” method. In the end, this depends on the proposed application, and any recommendation of a suitable pretreatment method must be based on a thorough techno-economic evaluation.

Efficient conversion of plant biomass to commodity chemicals is an important challenge that needs to be solved to enable a sustainable bioeconomy. Deconstruction of biomass to sugars and lignin yields a wide variety of low molecular weight carbon substrates that need to be funneled to product. Pseudomonas putida KT2440 has emerged as a potential platform for bioconversion of lignin and the other components of plant biomass. However, P. putida is unable to natively utilize several of the common sugars in hydrolysate streams, including galactose. In this work, we integrated a De Ley–Doudoroff catabolic pathway for galactose catabolism into the chromosome of P. putida KT2440, using genes from several different organisms. We found that the galactonate catabolic pathway alone (DgoKAD) supported slow growth of P. putida on galactose. Further integration of genes to convert galactose to galactonate and to optimize the transporter expression level resulted in a growth rate of 0.371 h−1. Additionally, the best-performing strain was demonstrated to co-utilize galactose with glucose. We have engineered P. putida to catabolize galactose, which will allow future engineered strains to convert more plant biomass carbon to products of interest. Further, by demonstrating co-utilization of glucose and galactose, continuous bioconversion processes for mixed sugar streams are now possible.

Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production. We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica. Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

Acyl-(acyl carrier protein (ACP)) reductase (AAR) is a key enzyme for hydrocarbon biosynthesis in cyanobacteria, reducing fatty acyl-ACPs to aldehydes, which are then converted into hydrocarbons by aldehyde-deformylating oxygenase (ADO). Previously, we compared AARs from various cyanobacteria and found that hydrocarbon yield in Escherichia coli coexpressing AAR and ADO was highest for AAR from Synechococcus elongatus PCC 7942 (7942AAR), which has high substrate affinity for 18-carbon fatty acyl-ACP, resulting in production of mainly heptadecene. In contrast, the hydrocarbon yield was lowest for AAR from Synechococcus sp. PCC 7336 (7336AAR), which has a high specificity for 16-carbon substrates, leading to production of mainly pentadecane. However, even the most productive AAR (7942AAR) still showed low activity; thus, residues within AAR that are nonconserved, but may still be important in hydrocarbon production need to be identified to engineer enzymes with improved hydrocarbon yields. Moreover, AAR mutants that favor shorter alkane production will be useful for producing diesel fuels with decreased freezing temperatures. Here, we aimed to identify such residues and design a highly productive and specific enzyme for hydrocarbon biosynthesis in E. coli. We introduced single amino acid substitutions into the least productive AAR (7336AAR) to make its amino acid sequence similar to that of the most productive enzyme (7942AAR). From the analysis of 41 mutants, we identified 6 mutations that increased either the activity or amount of soluble AAR, leading to a hydrocarbon yield improvement in E. coli coexpressing ADO. Moreover, by combining these mutations, we successfully created 7336AAR mutants with ~ 70-fold increased hydrocarbon production, especially for pentadecane, when compared with that of wild-type 7336AAR. Strikingly, the hydrocarbon yield was higher in the multiple mutants of 7336AAR than in 7942AAR. We successfully designed AAR mutants that, when coexpressed with ADO in E. coli, are more highly effective in hydrocarbon production, especially for pentadecane, than wild-type AARs. Our results provide a series of highly productive AARs with different substrate specificities, enabling the production of a variety of hydrocarbons in E. coli that may be used as biofuels.

Chromochloris zofingiensis is emerging as an industrially relevant alga given its robust growth for the production of lipids and astaxanthin, a value-added carotenoid with broad applications. Nevertheless, poor understanding of astaxanthin synthesis has limited engineering of this alga for rational improvements. To reveal the molecular mechanism underlying astaxanthin accumulation in C. zofingiensis, here we conducted an integrated analysis by combining the time-resolved transcriptomes and carotenoid profiling in response to nitrogen deprivation (ND). A global response was triggered for C. zofingiensis to cope with the ND stress. Albeit the little variation in total carotenoid content, individual carotenoids responded differentially to ND: the primary carotenoids particularly lutein and β-carotene decreased, while the secondary carotenoids increased considerably, with astaxanthin and canthaxanthin being the most increased ones. The carotenogenesis pathways were reconstructed: ND had little effect on the carbon flux to carotenoid precursors, but stimulated astaxanthin biosynthesis while repressing lutein biosynthesis, thereby diverting the carotenoid flux from primary carotenoids to secondary carotenoids particularly astaxanthin. Comparison between C. zofingiensis and Haematococcus pluvialis revealed the distinctive mechanism of astaxanthin synthesis in C. zofingiensis. Furthermore, potential bottlenecks in astaxanthin synthesis were identified and possible engineering strategies were proposed for the alga. Collectively, these findings shed light on distinctive mechanism of carotenogenesis for astaxanthin biosynthesis in C. zofingiensis, identify key functional enzymes and regulators with engineering potential and will benefit rational manipulation of this alga for improving nutritional traits.

Furfural and acetic acid are the two major inhibitors generated during lignocellulose pretreatment and hydrolysis, would severely inhibit the cell growth, metabolism, and ethanol fermentation efficiency of Zymomonas mobilis. Effective genome shuffling mediated by protoplast electrofusion was developed and then applied to Z. mobilis. After two rounds of genome shuffling, 10 different mutants with improved cell growth and ethanol yield in the presence of 5.0 g/L acetic acid and 3.0 g/L furfural were obtained. The two most prominent genome-shuffled strains, 532 and 533, were further investigated along with parental strains in the presence of 7.0 g/L acetic acid and 3.0 g/L furfural. The results showed that mutants 532 and 533 were superior to the parental strain AQ8-1 in the presence of 7.0 g/L acetic acid, with a shorter fermentation time (30 h) and higher productivity than AQ8-1. Mutant 533 exhibited subtle differences from parental strain F34 in the presence of 3.0 g/L furfural. Mutations present in 10 genome-shuffled strains were identified via whole-genome resequencing, and the source of each mutation was identified as either de novo mutation or recombination of the parent genes. These results indicate that genome shuffling is an efficient method for enhancing stress tolerance in Z. mobilis. The engineered strains generated in this study could be potential cellulosic ethanol producers in the future.

Anaerobic digestion of easily degradable biowaste can lead to the accumulation of volatile fatty acids, which will cause environmental stress to the sensitive methanogens consequently. The metabolic characteristics of methanogens under acetate stress can affect the overall performance of mixed consortia. Nevertheless, there exist huge gaps in understanding the responses of the dominant methanogens to the stress, e.g., Methanosarcinaceae. Such methanogens are resistant to environmental deterioration and able to utilize multiple carbon sources. In this study, transcriptomic and proteomic analyses were conducted to explore the responses of Methanosarcina barkeri strain MS at different acetate concentrations of 10, 25, and 50 mM. The trend of OD600 and the regulation of the specific genes in 50 mM acetate, indicated that high concentration of acetate promoted the acclimation of M. barkeri to acetate stress. Acetate stress hindered the regulation of quorum sensing and thereby eliminated the advantages of cell aggregation, which was beneficial to resist stress. Under acetate stress, M. barkeri allocated more resources to enhance the uptake of iron to maintain the integrities of electron-transport chains and other essential biological processes. Comparing with the initial stages of different acetate concentrations, most of the genes participating in acetoclastic methanogenesis did not show significantly different expressions except hdrB1C1, an electron-bifurcating heterodisulfide reductase participating in energy conversion and improving thermodynamic efficiency. Meanwhile, vnfDGHK and nifDHK participating in nitrogen fixation pathway were upregulated. In this work, transcriptomic and proteomic analyses are combined to reveal the responses of M. barkeri to acetate stress in terms of central metabolic pathways, which provides basic clues for exploring the responses of other specific methanogens under high organics load. Moreover, the results can also be used to gain insights into the complex interactions and geochemical cycles among natural or engineered populations. Furthermore, these findings also provide the potential for designing effective and robust anaerobic digesters with high organic loads.

Switchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration. Regenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety ‘Performer’ tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines. The new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6–7 months from culture establishment.

The original version of the article [1] unfortunately contained a mistake in author’s first name. The name of the author has been corrected from Wenqing Wang to Wenqin Wang in this correction article. The original article [1] has been corrected.

In the original version of the article [1], a calculation error resulted in a 3-order of magnitude mistake for the y-axis of the data reported in Fig. 5c and d.

Production of value-added materials from lignocellulosic biomass residues is an emerging sector that has attracted much attention as it offers numerous benefits from an environmental and economical point of view. Non-digestible oligosaccharides represent a group of carbohydrates that are resistant to gastrointestinal digestion, and therefore, they are considered as potential prebiotic candidates. Such oligosaccharides can derive from the biomass cellulose fraction through a controlled enzymatic hydrolysis that eliminates the yield of monomers. In the present study, hydrolysis of organosolv-pretreated forest residues (birch and spruce) was tested in the presence of four cellulases (EG5, CBH7, CBH6, EG7) and one accessory enzyme (LPMO). The optimal enzyme combinations were comprised of 20% EG5, 43% CBH7, 22% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of birch and 35% EG5, 45% CBH7, 10% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of spruce, leading to 22.3% and 19.1 wt% cellulose conversion into cellobiose, respectively. Enzymatic hydrolysis was applied on scale-up reactions, and the produced oligosaccharides (consisted of > 90% cellobiose) were recovered and separated from glucose through nanofiltration at optimized temperature (50 °C) and pressure (10 bar) conditions, yielding a final product with cellobiose-to-glucose ratio of 21.1 (birch) and 20.2 (spruce). Cellobiose-rich hydrolysates were tested as fermentative substrates for different lactic acid bacteria. It was shown that they can efficiently stimulate the growth of two Lactobacilli strains. Controlled enzymatic hydrolysis with processive cellulases, combined with product recovery and purification, as well as enzyme recycling can potentially support the sustainable production of food-grade oligosaccharides from forest biomass.

The development of renewable and sustainable biofuels to cover the future energy demand is one of the most challenging issues of our time. Biohydrogen, produced by photosynthetic microorganisms, has the potential to become a green biofuel and energy carrier for the future sustainable world, since it provides energy without CO2 emission. The recent development of two alternative protocols to induce hydrogen photoproduction in green algae enables the function of the O2-sensitive [FeFe]-hydrogenases, located at the acceptor side of photosystem I, to produce H2 for several days. These protocols prevent carbon fixation and redirect electrons toward H2 production. In the present work, we employed these protocols to a knockout Chlamydomonas reinhardtii mutant lacking flavodiiron proteins (FDPs), thus removing another possible electron competitor with H2 production. The deletion of the FDP electron sink resulted in the enhancement of H2 photoproduction relative to wild-type C. reinhardtii. Additionally, the lack of FDPs leads to a more effective obstruction of carbon fixation even under elongated light pulses. We demonstrated that the rather simple adjustment of cultivation conditions together with genetic manipulation of alternative electron pathways of photosynthesis results in efficient re-routing of electrons toward H2 photoproduction. Furthermore, the introduction of a short recovery phase by regular switching from H2 photoproduction to biomass accumulation phase allows to maintain cell fitness and use photosynthetic cells as long-term H2-producing biocatalysts.

The hydrotreatment of oleochemical/lipid feedstocks is currently the only technology that provides significant volumes (millions of litres per year) of “conventional” biojet/sustainable aviation fuels (SAF). However, if biojet fuels are to be produced in sustainably sourced volumes (billions of litres per year) at a price comparable with fossil jet fuel, biomass-derived “advanced” biojet fuels will be needed. Three direct thermochemical liquefaction technologies, fast pyrolysis, catalytic fast pyrolysis and hydrothermal liquefaction were assessed for their potential to produce “biocrudes” which were subsequently upgraded to drop-in biofuels by either dedicated hydrotreatment or co-processed hydrotreatment. A significant biojet fraction (between 20.8 and 36.6% of total upgraded fuel volume) was produced by all of the processes. When the fractions were assessed against general ASTM D7566 specifications they showed significant compliance, despite a lack of optimization in any of the process steps. When the life cycle analysis GHGenius model was used to assess the carbon intensity of the various products, significant emission reductions (up to 74%) could be achieved. It was apparent that the production of biojet fuels based on direct thermochemical liquefaction of biocrudes, followed by hydrotreating, has considerable potential.

l-Fucose is a rare sugar with potential uses in the pharmaceutical, cosmetic, and food industries. The enzymatic approach using l-fucose isomerase, which interconverts l-fucose and l-fuculose, can be an efficient way of producing l-fucose for industrial applications. Here, we performed biochemical and structural analyses of l-fucose isomerase identified from a novel species of Raoultella (RdFucI). RdFucI exhibited higher enzymatic activity for l-fuculose than for l-fucose, and the rate for the reverse reaction of converting l-fuculose to l-fucose was higher than that for the forward reaction of converting l-fucose to l-fuculose. In the equilibrium mixture, a much higher proportion of l-fucose (~ ninefold) was achieved at 30 °C and pH 7, indicating that the enzyme-catalyzed reaction favors the formation of l-fucose from l-fuculose. When biochemical analysis was conducted using l-fuculose as the substrate, the optimal conditions for RdFucI activity were determined to be 40 °C and pH 10. However, the equilibrium composition was not affected by reaction temperature in the range of 30 to 50 °C. Furthermore, RdFucI was found to be a metalloenzyme requiring Mn2+ as a cofactor. The comparative crystal structural analysis of RdFucI revealed the distinct conformation of α7–α8 loop of RdFucI. The loop is present at the entry of the substrate binding pocket and may affect the catalytic activity. RdFucI-catalyzed isomerization favored the reaction from l-fuculose to l-fucose. The biochemical and structural data of RdFucI will be helpful for the better understanding of the molecular mechanism of l-FucIs and the industrial production of l-fucose.

The availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media. An LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4–8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction. The biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent KM value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.

Xylanase is one of the most extensively used biocatalysts for biomass degradation. However, its low catalytic efficiency and poor thermostability limit its applications. Therefore, improving the properties of xylanases to enable synergistic degradation of lignocellulosic biomass with cellulase is of considerable significance in the field of bioenergy. Using fragment replacement, we improved the catalytic performance and thermostability of a GH10 xylanase, XylE. Of the ten hybrid enzymes obtained, seven showed xylanase activity. Substitution of fragments, M3, M6, M9, and their combinations enhanced the catalytic efficiency (by 2.4- to fourfold) as well as the specific activity (by 1.2- to 3.3-fold) of XylE. The hybrids, XylE-M3, XylE-M3/M6, XylE-M3/M9, and XylE-M3/M6/M9, showed enhanced thermostability, as observed by the increase in the T50 (3–4.7 °C) and Tm (1.1–4.7 °C), and extended t1/2 (by 1.8–2.3 h). In addition, the synergistic effect of the mutant xylanase and cellulase on the degradation of mulberry bark showed that treatment with both XylE-M3/M6 and cellulase exhibited the highest synergistic effect. In this case, the degree of synergy reached 1.3, and the reducing sugar production and dry matter reduction increased by 148% and 185%, respectively, compared to treatment with only cellulase. This study provides a successful strategy to improve the catalytic properties and thermostability of enzymes. We identified several xylanase candidates for applications in bioenergy and biorefinery. Synergistic degradation experiments elucidated a possible mechanism of cellulase inhibition by xylan and xylo-oligomers.

Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the Caldicellulosiruptor bescii CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. CbXyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target both xylan and cellulose. Intriguingly, the GH10 endoxylanase and GH48 cellobiohydrolase domains are both dual functional, raising the question if they can act synergistically in hydrolyzing cellulose and xylan, the two major components of plant cell wall. In this study, we discovered that CbXyn10C and CbCel48B, which stood for the N- and C-terminal catalytic domains, respectively, cooperatively released much more cellobiose and cellotriose from cellulose. In addition, they displayed intramolecular synergy but only at the early stage of xylan hydrolysis by generating higher amounts of xylooligosaccharides including xylotriose, xylotetraose, and xylobiose. When complex lignocellulose corn straw was used as the substrate, the synergy was found only for cellulose but not xylan hydrolysis. This is the first report to reveal the synergy between a GH10 and a GH48 domain. The synergy discovered in this study is helpful for understanding how C. bescii captures energy from these recalcitrant plant cell wall polysaccharides. The insight also sheds light on designing robust and multi-functional enzymes for plant cell wall polysaccharides degradation.

Lignin is the second most abundant natural polymer on earth. Industries using lignocellulosic biomass as feedstock generate a considerable amount of lignin as a byproduct with minimal usage. For a sustainable biorefinery, the lignin must be utilized in improved ways. Lignin is recalcitrant to degradation due to the complex and heterogeneous structure. The depolymerization of lignin and its conversion into specific product stream are the major challenges associated with lignin valorization. The blend of oligomeric, dimeric and monomeric lignin-derived compounds (LDCs) generated during depolymerization can be utilized by microbes for production of bioproducts. In the present study, a novel bacterium Burkholderia sp. strain ISTR5 (R5), a proteobacteria belonging to class betaproteobacteria, order Burkholderiales and family Burkholderiaceae, was isolated and characterized for the degradation of LDCs. R5 strain was cultured on 12 LDCs in mineral salt medium (MSM) supplemented with individual compounds such as syringic acid, p-coumaric acid, ferulic acid, vanillin, vanillic acid, guaiacol, 4-hydroxybenzoic acid, gallic acid, benzoic acid, syringaldehyde, veratryl alcohol and catechol. R5 was able to grow and utilize all the selected LDCs. The degradation of selected LDCs was monitored by bacterial growth, total organic carbon (TOC) removal and UV–Vis absorption spectra in scan mode. TOC reduction shown in the sample contains syringic acid 80.7%, ferulic acid 84.1%, p-coumaric acid 85.9% and benzoic acid 83.2%. In UV–Vis absorption spectral scan, most of the lignin-associated peaks were found at or near 280 nm wavelength in the obtained absorption spectra. Enzyme assay for the ligninolytic enzymes was also performed, and it was observed that lignin peroxidase and laccase were predominantly expressed. Furthermore, the GC–MS analysis of LDCs was performed to identify the degradation intermediates from these compounds. The genomic analysis showed the robustness of this strain and identified various candidate genes responsible for the degradation of aromatic or lignin derivatives, detoxification mechanism, oxidative stress response and fatty acid synthesis. The presence of peroxidases (13%), laccases (4%), monooxygenases (23%), dioxygenase (44%), NADPH: quinone oxidoreductases (16%) and many other related enzymes supported the degradation of LDCs. Numerous pathway intermediates were observed during experiment. Vanillin was found during growth on syringic acid, ferulic acid and p-coumaric acid. Some other intermediates like catechol, acetovanillone, syringaldehyde and 3,4-dihydroxybenzaldehyde from the recognized bacterial metabolic pathways existed during growth on the LDCs. The ortho- and meta cleavage pathway enzymes, such as the catechol-1,2-dioxygenase, protocatechuate 3,4-dioxygenase, catechol-2,3-dioxygenase and toluene-2,3-dioxygenase, were observed in the genome. In addition to the common aromatic degradation pathways, presence of the epoxyqueuosine reductase, 1,2-epoxyphenylacetyl-CoA isomerase in the genome advocates that this strain may follow the epoxy Coenzyme A thioester pathway for degradation.

For enzymes with buried active sites, transporting substrates/products ligands between active sites and bulk solvent via access tunnels is a key step in the catalytic cycle of these enzymes. Thus, tunnel engineering is becoming a powerful strategy to refine the catalytic properties of these enzymes. The tunnel-like structures have been described in enzymes catalyzing bulky substrates like glycosyl hydrolases, while it is still uncertain whether these structures involved in ligands exchange. Till so far, no studies have been reported on the application of tunnel engineering strategy for optimizing properties of enzymes catalyzing biopolymers. In this study, xylanase S7-xyl (PDB: 2UWF) with a deep active cleft was chosen as a study model to evaluate the functionalities of tunnel-like structures on the properties of biopolymer-degrading enzymes. Three tunnel-like structures in S7-xyl were identified and simultaneously reshaped through multi-sites saturated mutagenesis; the most advantageous mutant 254RL1 (V207N/Q238S/W241R) exhibited 340% increase in specific activity compared to S7-xyl. Deconvolution analysis revealed that all three mutations contributed synergistically to the improved activity of 254RL1. Enzymatic characterization showed that larger end products were released in 254RL1, while substrate binding and structural stability were not changed. Dissection of the structural alterations revealed that both the tun_1 and tun_2 in 254RL1 have larger bottleneck radius and shorter length than those of S7-xyl, suggesting that these tunnel-like structures may function as products transportation pathways. Attributed to the improved catalytic efficiency, 254RL1 represents a superior accessory enzyme to enhance the hydrolysis efficiency of cellulase towards different pretreated lignocellulose materials. In addition, tunnel engineering strategy was also successfully applied to improve the catalytic activities of three other xylanases including xylanase NG27-xyl from Bacillus sp. strain NG-27, TSAA1-xyl from Geobacillus sp. TSAA1 and N165-xyl from Bacillus sp. N16-5, with 80%, 20% and 170% increase in specific activity, respectively. This study represents a pilot study of engineering and functional verification of tunnel-like structures in enzymes catalyzing biopolymer. The specific activities of four xylanases with buried active sites were successfully improved by tunnel engineering. It is highly likely that tunnel reshaping can be used to engineer better biomass-degrading abilities in other lignocellulolytic enzymes with buried active sites.

The efficient utilization of lignocellulosic biomass for biofuel production has received increasing attention. Previous studies have investigated the pretreatment process of biomass, but the detailed enzymatic hydrolysis process of pretreated biomass remains largely unclear. Thus, this study investigated the pretreatment efficiency of dilute alkali, acid, hydrogen peroxide and its ultimate effects on enzymatic hydrolysis. Furthermore, to better understand the enzymatic digestion process of alkali-pretreated sweet sorghum straw (SSS), multimodal microscopy techniques were used to visualize the enzymatic hydrolysis process. After pretreatment with alkali, an enzymatic hydrolysis efficiency of 86.44% was obtained, which increased by 99.54% compared to the untreated straw (43.23%). The FTIR, XRD and SEM characterization revealed a sequence of microstructural changes occurring in plant cell walls after pretreatment, including the destruction of lignin–polysaccharide interactions, the increase of porosity and crystallinity, and reduction of recalcitrance. During the course of hydrolysis, the cellulase dissolved the cell walls in the same manner and the digestion firstly occurred from the middle of cell walls and then toward the cell wall corners. The CLSM coupled with fluorescent labeling demonstrated that the sclerenchyma cells and vascular bundles in natural SSS were highly lignified, which caused the nonproductive bindings of cellulase on lignin. However, the efficient delignification significantly increased the accessibility and digestibility of cellulase to biomass, thereby improving the saccharification efficiency. This work will be helpful in investigating the biomass pretreatment and its structural characterization. In addition, the visualization results of the enzymatic hydrolysis process of pretreated lignocellulose could be used for guidance to explore the lignocellulosic biomass processing and large-scale biofuel production.

Sorghum bicolor (L.) is an important bioenergy source. The stems of sweet sorghum function as carbon sinks and accumulate large amounts of sugars and lignocellulosic biomass and considerable amounts of starch, therefore providing a model of carbon allocation and accumulation for other bioenergy crops. While omics data sets for sugar accumulation have been reported in different genotypes, the common features of primary metabolism in sweet genotypes remain unclear. To obtain a cohesive and comparative picture of carbohydrate metabolism between sorghum genotypes, we compared the phenotypes and transcriptome dynamics of sugar-accumulating internodes among three different sweet genotypes (Della, Rio, and SIL-05) and two non-sweet genotypes (BTx406 and R9188). Field experiments showed that Della and Rio had similar dynamics and internode patterns of sugar concentration, albeit distinct other phenotypes. Interestingly, cellulose synthases for primary cell wall and key genes in starch synthesis and degradation were coordinately upregulated in sweet genotypes. Sweet sorghums maintained active monolignol biosynthesis compared to the non-sweet genotypes. Comparative RNA-seq results support the role of candidate Tonoplast Sugar Transporter gene (TST), but not the Sugars Will Eventually be Exported Transporter genes (SWEETs) in the different sugar accumulations between sweet and non-sweet genotypes. Comparisons of the expression dynamics of carbon metabolic genes across the RNA-seq data sets identify several candidate genes with contrasting expression patterns between sweet and non-sweet sorghum lines, including genes required for cellulose and monolignol synthesis (CesA, PTAL, and CCR), starch metabolism (AGPase, SS, SBE, and G6P-translocator SbGPT2), and sucrose metabolism and transport (TPP and TST2). The common transcriptome features of primary metabolism identified here suggest the metabolic networks contributing to carbon sink strength in sorghum internodes, prioritize the candidate genes for manipulating carbon allocation with bioenergy purposes, and provide a comparative and cohesive picture of the complexity of carbon sink strength in sorghum stem.

Fed-batch fermentation has been conventionally implemented for the production of lactic acid with a high titer and high productivity. However, its operation needs a complicated control which increases the production cost. This issue was addressed by simplifying the production scheme. Escherichia coli was manipulated for its glycerol dissimilation and d-lactate synthesis pathways and then subjected to adaptive evolution under high crude glycerol. Batch fermentation in the two-stage mode was performed by controlling the dissolved oxygen (DO), and the evolved strain deprived of poxB enabled production of 100 g/L d-lactate with productivity of 1.85 g/L/h. To increase productivity, the producer strain was further evolved to improve its growth rate on crude glycerol. The fermentation was performed to undergo the aerobic growth with low substrate, followed by the anaerobic production with high substrate. Moreover, the intracellular redox of the strain was balanced by fulfillment of the anaerobic respiratory chain with nitrate reduction. Without controlling the DO, the microbial fermentation resulted in the homofermentative production of d-lactate (ca. 0.97 g/g) with a titer of 115 g/L and productivity of 3.29 g/L/h. The proposed fermentation strategy achieves the highest yield based on crude glycerol and a comparable titer and productivity as compared to the approach by fed-batch fermentation. It holds a promise to sustain the continued development of the crude glycerol-based biorefinery.

For renewable and sustainable bioenergy utilization with cost-effectiveness, electron-shuttles (ESs) (or redox mediators (RMs)) act as electrochemical “catalysts” to enhance rates of redox reactions, catalytically accelerating electron transport efficiency for abiotic and biotic electrochemical reactions. ESs are popularly used in cellular respiratory systems, metabolisms in organisms, and widely applied to support global lives. Apparently, they are applicable to increase power-generating capabilities for energy utilization and/or fuel storage (i.e., dye-sensitized solar cell, batteries, and microbial fuel cells (MFCs)). This first-attempt review specifically deciphers the chemical structure association with characteristics of ESs, and discloses redox-mediating potentials of polyphenolics-abundant ESs via MFC modules. Moreover, to effectively convert electron-shuttling capabilities from non-sustainable antioxidant activities, environmental conditions to induce electrochemical mediation apparently play critical roles of great significance for bioenergy stimulation. For example, pH levels would significantly affect electrochemical potentials to be exhibited (e.g., alkaline pHs are electrochemically favorable for expression of such electron-shuttling characteristics). Regarding chemical structure effect, chemicals with ortho- and para-dihydroxyl substituents-bearing aromatics own convertible characteristics of non-renewable antioxidants and electrochemically catalytic ESs; however, ES capabilities of meta-dihydroxyl substituents can be evidently repressed due to lack of resonance effect in the structure for intermediate radical(s) during redox reaction. Moreover, this review provides conclusive remarks to elucidate the promising feasibility to identify whether such characteristics are non-renewable antioxidants or reversible ESs from natural polyphenols via cyclic voltammetry and MFC evaluation. Evidently, considering sustainable development, such electrochemically convertible polyphenolic species in plant extracts can be reversibly expressed for bioenergy-stimulating capabilities in MFCs under electrochemically favorable conditions.

Obtaining high-value products from lignocellulosic biomass is central for the realization of industrial biorefinery. Acid pretreatment has been reported to yield xylooligosaccharides (XOS) and improve enzymatic hydrolysis. Moreover, xylose, an inevitable byproduct, can be upgraded to xylonic acid (XA). The aim of this study was to valorize sugarcane bagasse (SB) by starting with XA pretreatment for XOS and glucose production within a multi-product biorefinery framework. SB was primarily subjected to XA pretreatment to maximize the XOS yield by the response surface method (RSM). A maximum XOS yield of 44.5% was achieved by acid pretreatment using 0.64 M XA for 42 min at 154 °C. Furthermore, XA pretreatment can efficiently improve enzymatic digestibility, and achieved a 90.8% cellulose conversion. In addition, xylose, the inevitable byproduct of the acid-hydrolysis of xylan, can be completely converted to XA via bio-oxidation of Gluconobacter oxydans (G. oxydans). Subsequently, XA and XOS can be simultaneously separated by electrodialysis. XA pretreatment was explored and exhibited a promising ability to depolymerize xylan into XOS. Mass balance analysis showed that the maximum XOS and fermentable sugars yields reached 10.5 g and 30.9 g per 100 g raw SB, respectively. In summary, by concurrently producing XOS and fermentable sugars with high yields, SB was thus valorized as a promising feedstock of lignocellulosic biorefinery for value-added products.

The recent discovery that LPMOs can work under anaerobic conditions when supplied with low amounts H2O2 opens the possibility of using LPMOs as enzyme aids in biogas reactors to increase methane yields from lignocellulosic materials. We have explored this possibility by studying anaerobic digestion of various lignocellulosic materials: Avicel, milled spruce and birch wood, and a lignin-rich hydrolysis residue from steam-exploded birch. The digestions were added LPMOs and various cellulolytic enzyme cocktails and were carried out with or without addition of H2O2. In several cases, enzyme addition had a beneficial effect on methane production, which was partly due to components present in the enzyme preparations. It was possible to detect LPMO activity during the initial phases of the anaerobic digestions of Avicel, and in some cases LPMO activity could be correlated with improved methane production from lignocellulosic materials. However, a positive effect on methane production was only seen when LPMOs were added together with cellulases, and never upon addition of LPMOs only. Generally, the experimental outcomes showed substrate-dependent variations in process efficiency and the importance of LPMOs and added H2O2. These differences could relate to variations in the type and content of lignin, which again will affect the activity of the LPMO, the fate of the added H2O2 and the generation of potentially damaging reactive-oxygen species. The observed effects showed that the interplay between cellulases and LPMOs is important for the overall efficiency of the process. This study shows that it may be possible to harness the power of LPMOs in anaerobic digestion processes and improve biogas production, but also highlight the complexity of the reaction systems at hand. One complicating factor was that the enzymes themselves and other organic components in the enzyme preparations acted as substrates for biogas production, meaning that good control reactions were essential to detect effects caused by enzyme activity. As also observed during regular aerobic enzymatic digestion of lignocellulosic biomass, the type and contents of lignin in the substrates likely plays a major role in determining the impact of LPMOs and of cellulolytic enzymes in general. More work is needed to unravel the interplay between LPMOs, O2, H2O2, and the multitude of redox-active components found in anaerobic bioreactors degrading lignocellulosic substrates.

Phenolic acids are lignin-derived fermentation inhibitors formed during many pretreatment processes of lignocellulosic biomass. In this study, vanillic, p-hydroxybenzoic, and syringic acids were selected as the model compounds of phenolic acids, and the effect of short-term adaptation strategies on the tolerance of S. cerevisiae to phenolic acids was investigated. The mechanism of phenolic acids tolerance in the adapted yeast strains was studied at the morphological and physiological levels. The multiple phenolic acids exerted the synergistic inhibitory effect on the yeast cell growth. In particular, a significant interaction between vanillic and hydroxybenzoic acids was found. The optimal short-term adaptation strategies could efficiently improve the growth and fermentation performance of the yeast strain not only in the synthetic media with phenolic acids, but also in the simultaneous saccharification and ethanol fermentation of corncob residue. Morphological analysis showed that phenolic acids caused the parental strain to generate many cytoplasmic membrane invaginations with crack at the top of these sites and some mitochondria gathered around. The adapted strain presented the thicker cell wall and membrane and smaller cell size than those of the parental strain. In particular, the cytoplasmic membrane generated many little protrusions with regular shape. The cytoplasmic membrane integrity was analyzed by testing the relative electrical conductivity, leakage of intracellular substance, and permeation of fluorescent probe. The results indicated that the short-term adaptation improved the membrane integrity of yeast cell. The inhibition mechanism of phenolic acid might be attributed to the combined effect of the cytoplasmic membrane damage and the intracellular acidification. The short-term adaptation strategy with varied stressors levels and adaptive processes accelerated the stress response of yeast cell structure to tolerate phenolic acids. This strategy will contribute to the development of robust microbials for biofuel production from lignocellulosic biomass.

β-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. In this study, BxlB—a highly secreted GH3 β-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites—was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants’ catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.

Cellulosic biomass, the earth’s most abundant renewable resource, can be used as substrates for biomanufacturing biofuels or biochemicals via in vitro synthetic enzymatic biosystems in which the first step is the enzymatic phosphorolysis of cellodextrin to glucose 1-phosphate (G1P) by cellodextrin phosphorylase (CDP). However, almost all the CDPs prefer cellodextrin synthesis to phosphorolysis, resulting in the low reaction rate of cellodextrin phosphorolysis for biomanufacturing. To increase the reaction rate of cellodextrin phosphorolysis, synthetic enzyme complexes containing CDP and phosphoglucomutase (PGM) were constructed to convert G1P to glucose 6-phosphate (G6P) rapidly, which is an important intermediate for biomanufacturing. Four self-assembled synthetic enzyme complexes were constructed with different spatial organizations based on the high-affinity and high-specific interaction between cohesins and dockerins from natural cellulosomes. Thus, the CDP–PGM enzyme complex with the highest enhancement of initial reaction rate was integrated into an in vitro synthetic enzymatic biosystem for generating bioelectricity from cellodextrin. The in vitro biosystem containing the best CDP–PGM enzyme complex exhibited a much higher current density (3.35-fold) and power density (2.14-fold) than its counterpart biosystem containing free CDP and PGM mixture. Hereby, we first reported bioelectricity generation from cellulosic biomass via in vitro synthetic enzymatic biosystems. This work provided a strategy of how to link non-energetically favorable reaction (cellodextrin phosphorolysis) and energetically favorable reaction (G1P to G6P) together to circumvent unfavorable reaction equilibrium and shed light on improving the reaction efficiency of in vitro synthetic enzymatic biosystems through the construction of synthetic enzyme complexes.

Hydrogen is considered a promising energy vector that can be produced from sustainable resources such as sunlight and water. In green algae, such as Chlamydomonas reinhardtii, photoproduction of hydrogen is catalyzed by the enzyme [FeFe]-hydrogenase (HydA). Although highly efficient, this process is transitory and thought to serve as a release valve for excess reducing power. Up to date, prolonged production of hydrogen was achieved by the deprivation of either nutrients or light, thus, hindering the full potential of photosynthetic hydrogen production. Previously we showed that the enzyme superoxide dismutase (SOD) can enhance HydA activity in vitro, specifically when tied together to a fusion protein. In this work, we explored the in vivo hydrogen production phenotype of HydA–SOD fusion. We found a sustained hydrogen production, which is dependent on linear electron flow, although other pathways feed it as well. In addition, other characteristics such as slower growth and oxygen production were also observed in Hyd–SOD-expressing algae. The Hyd–SOD fusion manages to outcompete the Calvin–Benson cycle, allowing sustained hydrogen production for up to 14 days in non-limiting conditions.

Efficient deconstruction of lignocellulosic biomass into simple sugars in an economically viable manner is a prerequisite for its global acceptance as a feedstock in bioethanol production. This is achieved in nature by suites of enzymes with the capability of efficiently depolymerizing all the components of lignocellulose. Here, we provide detailed insight into the repertoire of enzymes produced by microorganisms enriched from the gut of the crop pathogen rice yellow stem borer (Scirpophaga incertulas). A microbial community was enriched from the gut of the rice yellow stem borer for enhanced rice straw degradation by sub-culturing every 10 days, for 1 year, in minimal medium with rice straw as the main carbon source. The enriched culture demonstrated high cellulolytic and xylanolytic activity in the culture supernatant. Metatranscriptomic and metaexoproteomic analysis revealed a large array of enzymes potentially involved in rice straw deconstruction. The consortium was found to encode genes ascribed to all five classes of carbohydrate-active enzymes (GHs, GTs, CEs, PLs, and AAs), including carbohydrate-binding modules (CBMs), categorized in the carbohydrate-active enzymes (CAZy) database. The GHs were the most abundant class of CAZymes. Predicted enzymes from these CAZy classes have the potential to digest each cell-wall components of rice straw, i.e., cellulose, hemicellulose, pectin, callose, and lignin. Several identified CAZy proteins appeared novel, having an unknown or hypothetical catalytic counterpart with a known class of CBM. To validate the findings, one of the identified enzymes that belong to the GH10 family was functionally characterized. The enzyme expressed in E. coli efficiently hydrolyzed beechwood xylan, and pretreated and untreated rice straw. This is the first report describing the enrichment of lignocellulose degrading bacteria from the gut of the rice yellow stem borer to deconstruct rice straw, identifying a plethora of enzymes secreted by the microbial community when growing on rice straw as a carbon source. These enzymes could be important candidates for biorefineries to overcome the current bottlenecks in biomass processing.

Corn stover (CS) is evaluated as the most favorable candidate feedstock for butanol production via microbial acetone–butanol–ethanol (ABE) fermentation by Clostridium acetobutylicum. By independent acid pretreatment and enzymatic hydrolysis, fermentable sugars (mainly glucose and xylose) were released, of which glucose was naturally utilized as the most preferred carbon source by C. acetobutylicum. However, the ABE fermentation using corn stover hydrolysate (CSH) without detoxification is typically limited to poor sugars utilization, butanol production and productivity. In the presence of pretreatment-derived inhibitors, the intracellular ATP and NADH, as important factors involved in cell growth, solventogenesis initiation and stress response, are exceedingly challenged owing to disrupted glucose phosphotransferase system (PTS). Therefore, there is a necessity to develop effective engineering approaches to overcome these limitations for high-efficient butanol production from CSH without detoxification. PTS-engineered C. acetobutylicum strains were constructed via overexpression and knockout of gene glcG encoding glucose-specific PTS IICBA, which pleiotropically regulated glucose utilization, cell growth, solventogenesis and inhibitors tolerance. The PTSGlcG-overexpressing strain exhibited high fermentation efficiency, wherein butanol production and productivity was 11.1 g/L and 0.31 g/L/h, compared to those of 11.0 g/L and 0.15 g/L/h with the PTSGlcG-deficient strain. During CSH culture without detoxification, the PTSGlcG-overexpressing strain exhibited desirable inhibitors tolerance and solventogenesis with butanol production of 10.0 g/L, increased by 300% and 400% compared to those of 2.5 and 2.0 g/L with the control and PTSGlcG-deficient strains, respectively. As a result of extra glucose and 10 g/L CaCO3 addition into CSH, butanol production and productivity were further maximized to 12.5 g/L and 0.39 g/L/h. These validated improvements on the PTSGlcG-overexpressing strain were ascribed to not only efficient glucose transport but also its cascading effects on intracellular ATP and NADH generation, solventogenesis initiation and inhibitors tolerance at the exponential growth phase. The PTSGluG regulation could be an effective engineering approach for high-efficient ABE fermentation from lignocellulosic hydrolysates without detoxification or wastewater generation, providing fundamental information for economically sustainable butanol production with high productivity.

Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources. We metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture. We for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

Biobutanol has great potential as biofuel of the future. However, only a few organisms have the natural ability to produce butanol. Amongst them, Clostridium spp. are the most efficient producers. The high toxicity of biobutanol constitutes one of the bottlenecks within the biobutanol production process which often suffers from low final butanol concentrations and yields. Butanol tolerance is a key driver for process optimisation and, therefore, in the search for alternative butanol production hosts. Many Lactobacillus species show a remarkable tolerance to solvents and some Lactobacillus spp. are known to naturally produce 2-butanol from meso-2,3-butanediol (meso-2,3-BTD) during anaerobic sugar fermentations. Lactobacillus diolivorans showed already to be highly efficient in the production of other bulk chemicals using a simple two-step metabolic pathway. Exactly, the same pathway enables this cell factory for 2-butanol production. Due to the inability of L. diolivorans to produce meso-2,3-BTD, a two-step cultivation processes with Serratia marcescens has been developed. S. marcescens is a very efficient producer of meso-2,3-BTD from glucose. The process yielded a butanol concentration of 10 g/L relying on wild-type bacterial strains. A further improvement of the maximum butanol titer was achieved using an engineered L. diolivorans strain overexpressing the endogenous alcohol dehydrogenase pduQ. The two-step cultivation process based on the engineered strain led to a maximum 2-butanol titer of 13.4 g/L, which is an increase of 34%. In this study, L. diolivorans is for the first time described as a good natural producer for 2-butanol from meso-2,3-butanediol. Through the application of a two-step cultivation process with S. marcescens, 2-butanol can be produced from glucose in a one-vessel, two-step microbial process.

Itaconic acid is an unsaturated, dicarboxylic acid which finds a wide range of applications in the polymer industry and as a building block for fuels, solvents and pharmaceuticals. Currently, Aspergillus terreus is used for industrial production, with titers above 100 g L−1 depending on the conditions. Besides A. terreus, Ustilago maydis is also a promising itaconic acid production host due to its yeast-like morphology. Recent strain engineering efforts significantly increased the yield, titer and rate of production. In this study, itaconate production by U. maydis was further increased by integrated strain- and process engineering. Next-generation itaconate hyper-producing strains were generated using CRISPR/Cas9 and FLP/FRT genome editing tools for gene deletion, promoter replacement, and overexpression of genes. The handling and morphology of this engineered strain were improved by deletion of fuz7, which is part of a regulatory cascade that governs morphology and pathogenicity. These strain modifications enabled the development of an efficient fermentation process with in situ product crystallization with CaCO3. This integrated approach resulted in a maximum itaconate titer of 220 g L−1, with a total acid titer of 248 g L−1, which is a significant improvement compared to best published itaconate titers reached with U. maydis and with A. terreus. In this study, itaconic acid production could be enhanced significantly by morphological- and metabolic engineering in combination with process development, yielding the highest titer reported with any microorganism.

To produce second-generation biofuels, enzymatic catalysis is required to convert cellulose from lignocellulosic biomass into fermentable sugars. β-Glucosidases finalize the process by hydrolyzing cellobiose into glucose, so the efficiency of cellulose hydrolysis largely depends on the quantity and quality of these enzymes used during saccharification. Accordingly, to reduce biofuel production costs, new microbial strains are needed that can produce highly efficient enzymes on a large scale. We heterologously expressed the fungal β-glucosidase D2-BGL from a Taiwanese indigenous fungus Chaetomella raphigera in Pichia pastoris for constitutive production by fermentation. Recombinant D2-BGL presented significantly higher substrate affinity than the commercial β-glucosidase Novozyme 188 (N188; Km = 0.2 vs 2.14 mM for p-nitrophenyl β-d-glucopyranoside and 0.96 vs 2.38 mM for cellobiose). When combined with RUT-C30 cellulases, it hydrolyzed acid-pretreated lignocellulosic biomasses more efficiently than the commercial cellulase mixture CTec3. The extent of conversion from cellulose to glucose was 83% for sugarcane bagasse and 63% for rice straws. Compared to N188, use of D2-BGL halved the time necessary to produce maximal levels of ethanol by a semi-simultaneous saccharification and fermentation process. We upscaled production of recombinant D2-BGL to 33.6 U/mL within 15 days using a 1-ton bioreactor. Crystal structure analysis revealed that D2-BGL belongs to glycoside hydrolase (GH) family 3. Removing the N-glycosylation N68 or O-glycosylation T431 residues by site-directed mutagenesis negatively affected enzyme production in P. pastoris. The F256 substrate-binding residue in D2-BGL is located in a shorter loop surrounding the active site pocket relative to that of Aspergillus β-glucosidases, and this short loop is responsible for its high substrate affinity toward cellobiose. D2-BGL is an efficient supplement for lignocellulosic biomass saccharification, and we upscaled production of this enzyme using a 1-ton bioreactor. Enzyme production could be further improved using optimized fermentation, which could reduce biofuel production costs. Our structure analysis of D2-BGL offers new insights into GH3 β-glucosidases, which will be useful for strain improvements via a structure-based mutagenesis approach.

Waste lipids are attractive substrates for co-digestion with primary and activated sewage sludge (PASS) to improve biogas production at wastewater treatment plants. However, slow conversion rates of long-chain fatty acids (LCFA), produced during anaerobic digestion (AD), limit the applicability of waste lipids as co-substrates for PASS. Previous observations indicate that the sulfide level in PASS digesters affects the capacity of microbial communities to convert LCFA to biogas. This study assessed the microbial community response to LCFA loads in relation to sulfide level during AD of PASS by investigating process performance and microbial community dynamics upon addition of oleate (C18:1) and stearate (C18:0) to PASS digesters at ambient and elevated sulfide levels. Conversion of LCFA to biogas was limited (30% of theoretical biogas potential) during continuous co-digestion with PASS, which resulted in further LCFA accumulation. However, the accumulated LCFA were converted to biogas (up to 66% of theoretical biogas potential) during subsequent batch-mode digestion, performed without additional substrate load. Elevated sulfide level stimulated oleate (but not stearate) conversion to acetate, but oleate and sulfide imposed a synergistic limiting effect on acetoclastic methanogenesis and biogas formation. Next-generation sequencing of 16S rRNA gene amplicons of bacteria and archaea showed that differences in sulfide level and LCFA type resulted in microbial community alterations with distinctly different patterns. Taxonomic profiling of the sequencing data revealed that the phylum Cloacimonetes is likely a key group during LCFA degradation in PASS digesters, where different members take part in degradation of saturated and unsaturated LCFA; genus W5 (family Cloacimonadaceae) and family W27 (order Cloacimonadales), respectively. In addition, LCFA-degrading Syntrophomonas, which is commonly present in lipid-fed digesters, increased in relative abundance after addition of oleate at elevated sulfide level, but not without sulfide or after stearate addition. Stearate conversion to biogas was instead associated with increasing abundance of hydrogen-producing Smithella and hydrogenotrophic Methanobacterium. Long-chain fatty acid chain saturation and sulfide level are selective drivers for establishment of LCFA-degrading microbial communities in municipal sludge digesters.

Macroalgae and microalgae, as feedstocks for third-generation biofuel, possess competitive strengths in terms of cost, technology and economics. The most important compound in brown macroalgae is alginate, and the synergistic effect of endolytic and exolytic alginate lyases plays a crucial role in the saccharification process of transforming alginate into biofuel. However, there are few studies on the synergistic effect of endolytic and exolytic alginate lyases, especially those from the same bacterial strain. In this study, the endolytic alginate lyase AlyPB1 and exolytic alginate lyase AlyPB2 were identified from the marine bacterium Photobacterium sp. FC615. These two enzymes showed quite different and novel enzymatic properties whereas behaved a strong synergistic effect on the saccharification of alginate. Compared to that when AlyPB2 was used alone, the conversion rate of alginate polysaccharides to unsaturated monosaccharides when AlyPB1 and AlyPB2 acted on alginate together was dramatically increased approximately sevenfold. Furthermore, we found that AlyPB1 and AlyPB2 acted the synergistic effect basing on the complementarity of their substrate degradation patterns, particularly due to their M-/G-preference and substrate-size dependence. In addition, a novel method for sequencing alginate oligosaccharides was developed for the first time by combining the 1H NMR spectroscopy and the enzymatic digestion with the exo-lyase AlyPB2, and this method is much simpler than traditional methods based on one- and two-dimensional NMR spectroscopy. Using this strategy, the sequences of the final tetrasaccharide and pentasaccharide product fractions produced by AlyPB1 were easily determined: the tetrasaccharide fractions contained two structures, ΔGMM and ΔMMM, at a molar ratio of 1:3.2, and the pentasaccharide fractions contained four structures, ΔMMMM, ΔMGMM, ΔGMMM, and ΔGGMM, at a molar ratio of ~ 1:1.5:3.5:5.25. The identification of these two novel alginate lyases provides not only excellent candidate tool-type enzymes for oligosaccharide preparation but also a good model for studying the synergistic digestion and saccharification of alginate in biofuel production. The novel method for oligosaccharide sequencing described in this study will offer a very useful approach for structural and functional studies on alginate.

Euglena gracilis, a unicellular flagellated microalga, is regarded as one of the most promising species as microalgal feedstock for biofuels. Its lipids (mainly wax esters) are suitable for biodiesel and jet fuel. Culture of E. gracilis using wastewater effluent will improve the economics of E. gracilis biofuel production. Enhancement of the productivity of E. gracilis biomass is critical to creating a highly efficient biofuels production system. Certain bacteria have been found to promote microalgal growth by creating a favorable microenvironment. These bacteria have been characterized as microalgae growth-promoting bacteria (MGPB). Co-culture of microalgae with MGPB might offer an effective strategy to enhance microalgal biomass production in wastewater effluent culture systems. However, no MGPB has been identified to enhance the growth of E. gracilis. The objectives of this study were, therefore, to isolate and characterize the MGPB effective for E. gracilis and to demonstrate that the isolated MGPB indeed enhances the production of biomass and lipids by E. gracilis in wastewater effluent culture system. A bacterium, Emticicia sp. EG3, which is capable of promoting the growth of microalga E. gracilis, was isolated from an E. gracilis-municipal wastewater effluent culture. Biomass production rate of E. gracilis was enhanced 3.5-fold and 3.1-fold by EG3 in the co-culture system using a medium of heat-sterilized and non-sterilized wastewater effluent, respectively, compared to growth in the same effluent culture but without EG3. Two-step culture system was examined as follows: E. gracilis was cultured with or without EG3 in wastewater effluent in the first step and was further grown in wastewater effluent in the second step. Production yields of biomass and lipids by E. gracilis were enhanced 3.2-fold and 2.9-fold, respectively, in the second step of the system in which E. gracilis was co-cultured with EG3 in the first step. Emticicia sp. EG3 is the first MGPB for E. gracilis. Growth-promoting bacteria such as EG3 will be promising agents for enhancing E. gracilis biomass/biofuel productivities.

Due to its inevitable formation during biodiesel production and its relatively high degree of reduction, glycerol is an attractive carbon source for microbial fermentation processes. However, glycerol is catabolized in a fully respiratory manner by the eukaryotic platform organism Saccharomyces cerevisiae. We previously engineered S. cerevisiae strains to favor fermentative metabolism of glycerol by replacing the native FAD-dependent glycerol catabolic pathway with the NAD-dependent ‘DHA pathway’. In addition, a heterologous aquaglyceroporin (Fps1 homolog) was expressed to facilitate glycerol uptake. The current study was launched to scrutinize the formation of S. cerevisiae’s natural fermentation product ethanol from glycerol caused by the conducted genetic modifications. This understanding is supposed to facilitate future engineering of this yeast for fermenting glycerol into valuable products more reduced than ethanol. A strain solely exhibiting the glycerol catabolic pathway replacement produced ethanol at concentrations close to the detection limit. The expression of the heterologous aquaglyceroporin caused significant ethanol production (8.5 g L−1 from 51.5 g L−1 glycerol consumed) in a strain catabolizing glycerol via the DHA pathway but not in the wild-type background. A reduction of oxygen availability in the shake flask cultures further increased the ethanol titer up to 15.7 g L−1 (from 45 g L−1 glycerol consumed). The increased yield of cytosolic NADH caused by the glycerol catabolic pathway replacement seems to be a minimal requirement for the occurrence of alcoholic fermentation in S. cerevisiae growing in synthetic glycerol medium. The remarkable metabolic switch to ethanol formation in the DHA pathway strain with the heterologous aquaglyceroporin supports the assumption of a much stronger influx of glycerol accompanied by an increased rate of cytosolic NADH production via the DHA pathway. The fact that a reduction of oxygen supply increases ethanol production in DHA pathway strains is in line with the hypothesis that a major part of glycerol in normal shake flask cultures still enters the catabolism in a respiratory manner.