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2,3-Butanediol (2,3-BD) is a highly valued building block, and optimizing its production by fermentation, particularly with crude glycerol, is crucial. Enterobacter aerogenes is a key microorganism for this process; however, there are limited studies addressing the inhibition effects of products and by-products on 2,3-BD production. This study investigates these inhibition effects to maximize 2,3-BD production. Final concentrations of 2,3-BD plus acetoin reached 89.3, 92.7, and 71.1 g.L-1 with productivities of 1.22, 1.69, and 0.99 g.L-1.h-1 in pure glycerol, glucose, and crude glycerol media, respectively. Acetic acid was the main by-product, with concentrations ranging from 10 to 15 g.L-1. The reinoculation of E. aerogenes cells highlighted the strong effect of 2,3-BD and acetic acid on microbial growth and metabolism, with the cultivation environment exerting selective pressure. Notably, cells reuse enhanced performance in crude glycerol media, achieving a specific productivity in relation to biomass (YP/X) of 9.18 g.g-1; about 25% higher than in fed-batch without cells reuse. By combining results from two fed-batch cycles, the total final concentration of 2,3-BD plus acetoin reached 99.4 g.L-1, alongside a 33% reduction in total acetic acid production with reused cells.

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This study investigates the application of crude glycerol to the production of 1,3-propanediol by immobilized cells of Bacillus pumilus. This is a novel application of a naturally occurring producer obtained from a wastewater storage pond in Thailand. Crude glycerol was obtained through the methanolysis of palm oil, which was catalyzed using rice bran lipase. Ten components of the fermentation medium were screened using a Plackett-Burman design. The statistical significance of the results was determined using multiple linear regression with a backward elimination approach. The significance level was set to 5 % (p < 0.05). Only crude glycerol, (NH4)2SO4, MgSO4, and CaCl2 significantly affected 1,3-propanediol production by immobilized B. pumilus. Furthermore, preliminary screenings of environmental conditions used for 1,3-propanediol production were conducted using a Plackett-Burman design. The results showed that the temperature, time, and quantity of immobilized cells were factors that significantly affected 1,3-propanediol yield. Therefore, the quantities of crude glycerol, (NH4)2SO4, MgSO4, and CaCl2 and the temperature, time, and quantity of immobilized cells were optimized using response surface methodology based on a Box-Behnken design. The model predicted a maximum 1,3-propanediol yield of 45.68 g/L with the following conditions: 60 g/L crude glycerol, 5 g/L (NH4)2SO4, 0.55 g/L MgSO4, 0.05 g/L CaCl2, a fermentation duration of 101 h, and a temperature of 25 °C, with 250 g of immobilized cells. The validation trials confirmed a production level of 44.12 ± 1.81 g/L, indicating a 2.86-fold production increase relative to the control group. Overall, this study demonstrates the potential of using crude glycerol as a substrate to improve the yields of 1,3-propanediol produced by B. pumilus.

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BACKGROUND: (R,R)-2,3-butanediol (BDO) is employed in a variety of applications and is gaining prominence due to its unique physicochemical features. The use of glycerol as a carbon source for 2,3-BDO production in Klebsiella pneumoniae has been limited, since 1,3-propanediol (PDO) is generated during glycerol fermentation. RESULTS: In this study, the inactivation of the budC gene in K. pneumoniae increased the production rate of (R,R)-2,3-BDO from 21.92 ± 2.10 to 92.05 ± 1.20%. The major isomer form of K. pneumoniae (meso-2,3-BDO) was shifted to (R,R)-2,3-BDO. The purity of (R,R)-2,3-BDO was examined by agitation speed, and 98.54% of (R,R)-2,3-BDO was obtained at 500 rpm. However, as the cultivation period got longer, the purity of (R,R)-2,3-BDO declined. For this problem, a two-step agitation speed control strategy (adjusted from 500 to 400 rpm after 24 h) and over-expression of the dhaD gene involved in (R,R)-2,3-BDO biosynthesis were used. Nevertheless, the purity of (R,R)-2,3-BDO still gradually decreased over time. Finally, when pure glycerol was replaced with crude glycerol, the titer of 89.47 g/L of (R,R)-2,3-BDO (1.69 g/L of meso-2,3-BDO), productivity of 1.24 g/L/h, and yield of 0.35 g/g consumed crude glycerol was achieved while maintaining a purity of 98% or higher. CONCLUSIONS: This study is meaningful in that it demonstrated the highest production and productivity among studies in that produced (R,R)-2,3-BDO with a high purity in Klebsiella sp. strains. In addition, to the best of our knowledge, this is the first study to produce (R,R)-2,3-BDO using glycerol as the sole carbon source.

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The strain Gluconobacter oxydans LMG 1385 was used for the bioconversion of crude glycerol to dihydroxyacetone. The suitability of fed-batch cultures for the production of dihydroxyacetone was determined, and the influence of the pH of the culture medium and the initial concentration of glycerol on maximizing the concentration of dihydroxyacetone and on the yield and speed of obtaining dihydroxyacetone by bioconversion was examined. The feeding strategy of the substrate (crude glycerol) during the process was based on measuring the dissolved oxygen tension of the culture medium. The highest concentration of dihydroxyacetone PK = 175.8 g·L-1 and the highest yield YP/Sw = 94.3% were obtained when the initial concentration of crude glycerol was S0 = 70.0 g·L-1 and the pH of the substrate was maintained during the process at level 5.0.

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The growth of the biodiesel industry has resulted in significant quantity of crude glycerol. It is necessary to explore the synthesis of high-value-added products from crude glycerol. In this study, the enzymatic synthesis of monoglycerides under solvent-free conditions, employing crude glycerol as the primary feedstock, had been investigated. The analysis showed that the highest yield of monoglycerides was obtained after 12 h, and Novozym 435 showed the highest monoglyceride yield of 18.41 % among the three lipases tested, followed by Lipozyme TL IM and Lipozyme RM IM. Monoglycerides were synthesized from biodiesel-derived crude glycerol using Novozym 435 as the catalyst under solvent-free conditions at different parameters, which were catalyst concentration, substrate molar ratio, and temperature. The yield of monoglycerides was examined in single-factor experiments. Response surface methodology (RSM) was subsequently employed to optimize the synthesis conditions based on the single-factor experimental results. The optimal conditions were at an enzyme concentration of 12.7 wt% and a molar ratio of crude glycerol:oil of 5.7:1 at a reaction temperature of 65.2 °C. The experimental yield of monoglycerides under the optimal conditions was 28.93 %, which is close to the value predicted from the RSM model (29.02 %).

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BACKGROUND: Glycerol, as a by-product, mainly derives from the conversion of many crops to biodiesel, ethanol, and fatty ester. Its bioconversion to 1,3-propanediol (1,3-PDO) is an environmentally friendly method. Continuous fermentation has many striking merits over fed-batch and batch fermentation, such as high product concentration with easy feeding operation, long-term high productivity without frequent seed culture, and energy-intensive sterilization. However, it is usually difficult to harvest high product concentrations. RESULTS: In this study, a three-stage continuous fermentation was firstly designed to produce 1,3-PDO from crude glycerol by Clostridium butyricum, in which the first stage fermentation was responsible for providing the excellent cells in a robust growth state, the second stage focused on promoting 1,3-PDO production, and the third stage aimed to further boost the 1,3-PDO concentration and reduce the residual glycerol concentration as much as possible. Through the three-stage continuous fermentation, 80.05 g/L 1,3-PDO as the maximum concentration was produced while maintaining residual glycerol of 5.87 g/L, achieving a yield of 0.48 g/g and a productivity of 3.67 g/(L·h). Based on the 14 sets of experimental data from the first stage, a kinetic model was developed to describe the intricate relationships among the concentrations of 1,3-PDO, substrate, biomass, and butyrate. Subsequently, this kinetic model was used to optimize and predict the highest 1,3-PDO productivity of 11.26 g/(L·h) in the first stage fermentation, while the glycerol feeding concentration and dilution rate were determined to be 92 g/L and 0.341 h-1, separately. Additionally, to achieve a target 1,3-PDO production of 80 g/L without the third stage fermentation, the predicted minimum volume ratio of the second fermenter to the first one was 11.9. The kinetics-based two-stage continuous fermentation was experimentally verified well with the predicted results. CONCLUSION: A novel three-stage continuous fermentation and a kinetic model were reported. Then a simpler two-stage continuous fermentation was developed based on the optimization of the kinetic model. This kinetics-based development of two-stage continuous fermentation could achieve high-level production of 1,3-PDO. Meanwhile, it provides a reference for other bio-chemicals production by applying kinetics to optimize multi-stage continuous fermentation.

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Biodiesel production produces significant quantities of impure crude glycerol as a by-product. Recent increases in the global biodiesel production have led to a surplus of crude glycerol, rendering it a waste. As a result, different methods for its valorisation are currently being investigated. This paper assesses the life cycle environmental impacts of an emerging technology for purification of crude glycerol - a multi-step physico-chemical treatment - in comparison to incineration with energy recovery commonly used for its disposal. For the former, three different acids (H3PO4, H2SO4 and HCl) are considered for the acidification step in the purification process. The results suggest that the H2SO4-based treatment is the best option with 17 net-negative impacts out of the 18 categories considered; this is due to system credits for the production of purified glycerol, heat and potassium salts. In comparison to incineration with energy recovery, the H2SO4-based process has lower savings for the climate change impact (-311 versus -504 kg CO2 eq./t crude glycerol) but it performs better in ten other categories. Sensitivity analyses suggest that that the impacts of the physico-chemical treatment are highly dependent on crude glycerol composition, allocation of burdens to crude glycerol and credits for glycerol production. For example, treating crude glycerol with lower glycerol content would increase all impacts except climate change and fossil depletion due to the higher consumption of chemicals and lower production of purified glycerol. Considering crude glycerol as a useful product rather than waste and allocating to it burdens from biodiesel production would increase most impacts significantly, including climate change (22-40 %), while fossil depletion, freshwater and marine eutrophication would become net-positive. The findings of this research will be of interest to the biodiesel industry and other industrial sectors that generate crude glycerol as a by-product.

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With the rapid development of biodiesel, biodiesel-derived glycerol has become a promising renewable bioresource. The key to utilizing this bioresource lies in the value-added conversion of crude glycerol. While purifying crude glycerol into a pure form allows for diverse applications, the intricate nature of this process renders it costly and environmentally stressful. Consequently, technology facilitating the direct utilization of unpurified crude glycerol holds significant importance. It has been reported that crude glycerol can be bio-transformed or chemically converted into high-value polymers. These technologies provide cost-effective alternatives for polymer production while contributing to a more sustainable biodiesel industry. This review article describes the global production and quality characteristics of biodiesel-derived glycerol and investigates the influencing factors and treatment of the composition of crude glycerol including water, methanol, soap, matter organic non-glycerol, and ash. Additionally, this review also focused on the advantages and challenges of various technologies for converting crude glycerol into polymers, considering factors such as the compatibility of crude glycerol and the control of unfavorable factors. Lastly, the application prospect and value of crude glycerol conversion were discussed from the aspects of economy and environmental protection. The development of new technologies for the increased use of crude glycerol as a renewable feedstock for polymer production will be facilitated by the findings of this review, while promoting mass market applications.

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Polyhydroxyalkanoates (PHA) recovery from industrial wastewater has been highlighted as a promising strategy for a circular bioeconomy. However, the high and varying level of nitrogen in wastewater makes enrichment of mixed microbial culture (MMC) low efficiency. In this study, spatial separation of nitrifiers and denitrifiers was adopted by adding biocarriers in MMC and decreasing the sludge retention time (SRT) to accelerate the enrichment of PHA-storing MMC fed by mixed wastewater containing glycerol and propionate. Nitrifiers and denitrifiers were sustained on biocarriers, obtaining a high total inorganic nitrogen removal and allowing a more efficient selective pressure of a high carbon and nitrogen ratio (C/N) under low SRT conditions. The maximum PHA cell content and relative abundance of PHA-storing bacteria were increased to 60.51 % (SRT 6 d) and 49.62 % (SRT 6 d) with the decrease of SRT, respectively. This study demonstrates an efficient way to highly enrich PHA-storing MMC from crude glycerol, which provide a relevant technical support for high-efficiency enrichment of PHA-storing bacteria in low C/N wastewater.

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Rhodotorula paludigena CM33 is an oleaginous yeast that has been demonstrated to accumulate substantial quantities of intracellular lipids and carotenoids. In this study, crude glycerol, a by-product of biodiesel production, was used as a carbon source to enhance the accumulation of lipids and carotenoids in the cells. The culture conditions were first optimized using response surface methodology, which revealed that the carotenoid concentration and lipid content improved when the concentration of crude glycerol was 40 g/L. Different fermentation conditions were also investigated: batch, repeated-batch, and fed-batch conditions in a 500 L fermenter. For fed-batch fermentation, the maximum concentrations of biomass, lipids, and carotenoids obtained were 46.32 g/L, 37.65%, and 713.80 mg/L, respectively. A chemical-free carotenoid extraction method was also optimized using high-pressure homogenization and a microfluidizer device. The carotenoids were found to be mostly beta-carotene, which was confirmed by HPLC (high pressure liquid chromatography), LC-MS (liquid chromatography-mass spectrometry), and NMR (nuclear magnetic resonance). The results of this study indicate that crude glycerol can be used as a substrate to produce carotenoids, resulting in enhanced value of this biodiesel by-product.

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BACKGROUND: The direct bioconversion of crude glycerol, a byproduct of biodiesel production, into 1,3-propanediol by microbial fermentation constitutes a remarkably promising value-added applications. However, the low activity of glycerol dehydratase, which is the key and rate-limiting enzyme in the 1,3-propanediol synthetic pathway, caused by crude glycerol impurities is one of the main factors affecting the 1,3-propanediol yield. Hence, the exploration of glycerol dehydratase resources suitable for crude glycerol bioconversion is required for the development of 1,3-propanediol-producing engineered strains. RESULTS: In this study, the novel glycerol dehydratase 2eGDHt, which has a tolerance against crude glycerol impurities from Klebsiella pneumoniae 2e, was characterized. The 2eGDHt exhibited the highest activity toward glycerol, with Km and Vm values of 3.42 mM and 58.15 nkat mg-1, respectively. The optimum pH and temperature for 2eGDHt were 7.0 and 37 °C, respectively. 2eGDHt displayed broader pH stability than other reported glycerol dehydratases. Its enzymatic activity was increased by Fe2+ and Tween-20, with 294% and 290% relative activities, respectively. The presence of various concentrations of the crude glycerol impurities, including NaCl, methanol, oleic acid, and linoleic acid, showed limited impact on the 2eGDHt activity. In addition, the enzyme activity was almost unaffected by the presence of an impurity mixture that mimicked the crude glycerol environment. Structural analyses revealed that 2eGDHt possesses more coil structures than reported glycerol dehydratases. Moreover, molecular dynamics simulations and site-directed mutagenesis analyses implied that the existence of unique Val744 from one of the increased coil regions played a key role in the tolerance characteristic by increasing the protein flexibility. CONCLUSIONS: This study provides insight into the mechanism for enzymatic action and the tolerance against crude glycerol impurities, of a novel glycerol dehydratase 2eGDHt, which is a promising glycerol dehydratase candidate for biotechnological conversion of crude glycerol into 1,3-PDO.

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Polyhydroxybutyrate (PHB) is an attractive biodegradable polymer that can be produced through the microbial fermentation of organic wastes or wastewater. However, its mass production has been restricted by the poor utilization of organic wastes due to the presence of inhibitory substances, slow microbial growth, and high energy input required for feedstock sterilization. Here, Vibrio natriegens, a fast-growing bacterium with a broad substrate spectrum and high tolerance to salt and toxic substances, was genetically engineered to enable efficient PHB production from nonsterilized fermentation of organic wastes. The key genes encoding the PHB biosynthesis pathway of V. natriegens were identified through base editing and overexpressed. The metabolically engineered strain showed 166-fold higher PHB content (34.95 wt %) than the wide type when using glycerol as a substrate. Enhanced PHB production was also achieved when other sugars were used as feedstock. Importantly, it outperformed the engineered Escherichia coli MG1655 in PHB productivity (0.053 g/L/h) and tolerance to toxic substances in crude glycerol, without obvious activity decline under nonsterilized fermentation conditions. Our work demonstrates the great potential of engineered V. natriegens for low-cost PHB bioproduction and lays a foundation for exploiting this strain as a next-generation model chassis microorganism in synthetic biology.

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Renewable hydrogen production by aqueous phase reforming (APR) over Ni/Al-Ca catalysts was studied using pure or refined crude glycerol as feedstock. The APR was carried out in a fixed bed reactor at 238 °C, 37 absolute bar for 3 h, using a solution of 5 wt.% of glycerol, obtaining gas and liquid products. The catalysts were prepared by the co-precipitation method, calcined at different temperatures, and characterized before and after their use by several techniques (XRD, ICP-OES, H2-TPR, NH3-TPD, CO2-TPD, FESEM, and N2-physisorption). Increasing the calcination temperature and adding Ca decreased the surface area from 256 to 188 m2/g, and its value after the APR changed depending on the feedstock used. The properties of the acid and basic sites of the catalysts influenced the H2 yield also depending on the feed used. The Ni crystallite was between 6 and 20 nm. In general, the incorporation of Ca into Ni-based catalysts and the increase of the calcination temperature improved H2 production, obtaining 188 mg H2/mol C fed during the APR of refined crude glycerol over Ni/AlCa-675 catalyst, which was calcined at 675 °C. This is a promising result from the point of view of enhancing the economic viability of biodiesel.

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This study aimed to examine the impact of crude glycerol as the main carbon source on the growth, cell morphology, and production of high-value-added metabolites of two microalgal species, namely Chlorella vulgaris and Scenedesmus quadricauda, under heterotrophic and mixotrophic conditions, using monochromatic illumination from light-emitting diodes (LEDs) emitting blue, red, yellow, and white (control) light. The findings indicated that both microalgae strains exhibited higher biomass yield on the mixotrophic growth system when compared to the heterotrophic one, while S. quadricauda generally performed better than C. vulgaris. In mixotrophic mode, the use of different monochromatic illumination affected biomass production differently on both strains. In S. quadricauda, growth rate was higher under red light (µmax = 0.89 d-1), while the highest biomass concentration and yield per gram of consumed glycerol were achieved under yellow light, reaching 1.86 g/L and Yx/s = 0.18, respectively. On the other hand, C. vulgaris demonstrated a higher growth rate on blue light (µmax = 0.45 d-1) and a higher biomass production on white (control) lighting (1.34 g/L). Regarding the production of metabolites, higher yields were achieved during mixotrophic mode in both strains. In C. vulgaris, the highest lipid (26.5% of dry cell weight), protein (63%), and carbohydrate (20.3%) contents were obtained under blue, red, and yellow light, respectively, thus indicating that different light wavelengths probably activate different metabolic pathways. Similar results were obtained for S. quadricauda with red light leading to higher lipid content, while white lighting caused higher production of proteins and carbohydrates. Overall, the study demonstrated the potential of utilizing crude glycerol as a carbon source for the growth and metabolite production of microalgae and, furthermore, revealed that the strains' behavior varied depending on lighting conditions.

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Bio-polyols, produced by liquefying lignin with polyhydric alcohols, offer a promising alternative to conventional polyols for polyurethane production. To enhance the sustainability on the production of these bio-polyols, this study proposes the use of crude glycerol and microwave-assisted liquefaction as substitutes for conventional methods and commercial glycerol. This approach reduces the energy requirements of the reaction while also adding value to this by-product. The synthesis of bio-polyols with suitable properties to produce elastic and rigid polyurethane was carried out using previously optimised reaction conditions. Organosolv lignins obtained from Eucalyptus globulus and Pinus radiata were employed, using polyethylene glycol and crude glycerol as solvents and sulphuric acid as a catalyst. Several parameters of the bio-polyols were analysed, including hydroxyl number (IOH), acid number (An), and functionality (f), suggesting that the bio-polyols were suitable for polyurethane synthesis. Bio-polyols formulated to produce rigid polyurethanes exhibited IOH values of 554 and 383 (mg KOH/g), An values of 1.91 and 4.21 (mg KOH/g), and functionalities of 4.16 and 3.14 for Eucalyptus globulus and Pinus radiata lignin. In the case of bio-polyols for elastic polyurethanes, the values were 228 and 173 (mg KOH/g) (IOH), 20.94 and 25.09 (mg KOH/g) (An), and functionalities of 3.51 and 2.08.

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Poly(3-hydroxybutyrate) (PHB) is a prominent bio-plastic and recognized as the potential replacement of petroleum-derived plastics. To make PHB cost-effective, the production scheme based on crude glycerol was developed using Escherichia coli. The heterogeneous synthesis pathway of PHB was introduced into the E. coli strain capable of efficiently utilizing glycerol. The central metabolism that links to the synthesis of acetyl-CoA and NADPH was further reprogrammed to improve the PHB production. Key genes were targeted for manipulation, involving those in glycolysis, the pentose phosphate pathway, and the tricarboxylic cycle. As a result, the engineered strain gained a 22-fold increase in the PHB titer. Finally, the fed-batch fermentation was conducted with the producer strain to give the PHB titer, content, and productivity reaching 36.3 ± 3.0 g/L, 66.5 ± 2.8%, and 1.2 ± 0.1 g/L/h, respectively. The PHB yield on crude glycerol accounts for 0.3 g/g. The result indicates that the technology platform as developed is promising for the production of bio-plastics.

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Crude glycerol (CG) and glycerol pitch (GP) are highly alkaline residues from biodiesel and oleochemical plants, respectively, and have organic content which incurs high disposal cost and poses an environmental threat. Characterization of these residues for composition and properties could provide insight into their quality for proper disposal and can help the biodiesel industry to adopt more sustainable practices, such as reducing waste and improving the efficiency of the production process, hence minimizing the impact of the biodiesel supply chain to the environment. These data also allow the identification and exploration of new ways for their utilization and transformation into highly value-added products. In this study, we evaluated four CG samples (B, C, D, and E) and two GP samples (F and G) obtained from Malaysian palm oil refineries, and the results were compared with pure glycerol (A). Spectroscopic analysis was performed using FTIR, 1H-, and 13C-NMR. All samples had similar density to A (1.26 g/cm3), except for F (1.31 g/cm3), while the density for E and G could not be determined due to their physical states. The pH and viscosity largely varied in the range of 7.26-11.89 and 43-225 cSt, respectively. The glycerol content of CG (B, C, D, and E) was high and consistent (81.7-87.3%) whereas GP F and G had 71.5 and 63.9% glycerol content, respectively. Major contaminants in CG and GP were water and matter organic non-glycerol (MONG), respectively. The water, ash, soap, and salt content were considerably low, which varied from 3.4 to 14.1%, 3.9 to 13.0%, 0.1 to 5.7%, and 4.1 to 9.2% respectively. Thermal analysis of CG and GP exhibited four phases of decomposition attributed to the impurities compared to the single phase in A. All samples had calorific values lower than A (18.1 MJ/kg) between 9.0 and 17.7 MJ/kg. Based on the results, CG and GP have high glycerol content which reveals their potential to be used as feedstock in bioconversion and chemical or thermal treatment while impurities may be removed by pre-treatment if required. As palm oil is one of the main feedstocks for the oleochemical industry, this work underlines the importance of characterization of the residue generated to provide additional data and information on palm-based agricultural industry wastes, minimize the impact of palm oil supply chain on the environment, and explore its potential usage for value-addition. Supplementary Information: The online version contains supplementary material available at 10.1007/s13399-023-04003-4.

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Saccharification is a pivotal step in the conversion of lignocellulose to biofuels and chemicals. In this study, crude glycerol derived from biodiesel production was used in pretreatment to facilitate efficient and clean pyrolytic saccharification of sugarcane bagasse. Delignification, demineralization, destruction of lignin-carbohydrate complex structure, and cellulose crystallinity improvement in crude glycerol pretreated biomass could enhance levoglucosan producing reactions against competitive reactions, and therefore facilitate a kinetically controlled pyrolysis with apparent activation energy increased by 2-fold. Accordingly, selective levoglucosan production (44.4%) was promoted by 6-fold whilst light oxygenates and lignin monomers were limited to <25% in bio-oil. Owing to the high-efficiency saccharification, life cycle assessment suggested that the environmental impacts of the integrated process were less than those of typical acid pretreatment and petroleum-based processes, especially on the acidification (8-fold less) and global warming potential. This study provides an environmentally benign approach to efficient biorefinery and waste management.

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BACKGROUND: Yarrowia lipolytica is a well-studied oleaginous yeast known for its ability to accumulate and store intracellular lipids, while growing on diverse, non-conventional substrates. Amongst them, crude glycerol, a low-cost by-product of the biodiesel industry, appears to be an interesting option for scaling up a sustainable single-cell oil production process. Adaptive laboratory evolution (ALE) is a powerful tool to force metabolic adaptations endowing tolerance to stressful environmental conditions, generating superior phenotypes with industrial relevance. RESULTS: Y. lipolytica MUCL 28849 underwent ALE in a synthetic medium with increasing concentration of pure or crude glycerol as a stressing factor (9-20% v/v) for 520 generations. In one case of pure glycerol, chemical mutagenesis with ethyl methanesulfonate (EMS) was applied prior to ALE. Growth profile, biomass production and lipid content of 660 evolved strains (EVS), revealed 5 superior isolates; exhibiting from 1.9 to 3.6-fold increase of dry biomass and from 1.1 to 1.6-fold increase of lipid concentration compared to the parental strain, when grown in 15% v/v crude glycerol. NGS for differential gene expression analysis, showed induced expression in all EVS affecting nucleosomal structure and regulation of transcription. As strains differentiated, further changes accumulated in membrane transport and protein transport processes. Genes involved in glycerol catabolism and triacylglycerol biosynthesis were overexpressed in two EVS. Mismatches and gaps in the expressed sequences identified altered splicing and mutations in the EVS, with most of them, affecting different components of septin ring formation in the budding process. The selected YLE155 EVS, used for scale-up cultivation in a 3L benchtop bioreactor with 20% v/v crude glycerol, achieved extended exponential phase, twofold increase of dry biomass and lipid yields at 48 h, while citric acid secretion and glycerol consumption rates were 40% and 50% lower, respectively, compared to the parental strain, after 24 h of cultivation. CONCLUSION: ALE and EMS-ALE under increasing concentrations of pure or crude glycerol generated novel Y. lipolytica strains with enhanced biomass and lipid content. Differential gene expression analysis and scale-up of YLE155, illustrated the potential of the evolved strains to serve as suitable "chassis" for rational engineering approaches towards both increased lipid accumulation, and production of high-added value compounds, through efficient utilization of crude glycerol.

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Petrochemical-based plastics are hardly biodegradable and a major cause of environmental pollution, and polyhydroxybutyrate (PHB) is attracting attention as an alternative due to its similar properties. However, the cost of PHB production is high and is considered the greatest challenge for its industrialization. Here, crude glycerol was used as a carbon source for more efficient PHB production. Among the 18 strains investigated, Halomonas taeanenisis YLGW01 was selected for PHB production due to its salt tolerance and high glycerol consumption rate. Furthermore, this strain can produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3 HV)) with 17 % 3 HV mol fraction when a precursor is added. PHB production was maximized through medium optimization and activated carbon treatment of crude glycerol, resulting in 10.5 g/L of PHB with 60 % PHB content in fed-batch fermentation. Physical properties of the produced PHB were analyzed, i.e., weight average molecular weight (6.8 × 105), number average molecular weight (4.4 × 105), and the polydispersity index (1.53). In the universal testing machine analysis, the extracted intracellular PHB showed a decrease in Young's modulus, an increase in Elongation at break, greater flexibility than authentic film, and decreased brittleness. This study confirmed that YLGW01 is a promising strain for industrial PHB production using crude glycerol.

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