Efficient recovery of long-chain dicarboxylic acids (LCDAs) from aqueous fermentation broths is a key challenge for the industrial development of bio-based LCDA production. This study evaluates liquid–liquid extraction (LLE) as a downstream recovery strategy, comparing physical extraction (PE) and reactive extraction (RE) for DCA 12, DCA 16, and DCA 18. The novelty of this work lies in demonstrating that LCDA extraction is governed by mechanisms fundamentally different from those of short- and medium-chain dicarboxylic acids. Whereas shorter chain dicarboxylic acids are mainly controlled by dissociation degree, LCDA recovery is strongly influenced by carbon-chain apolarity, low aqueous solubility, and compound losses through agglomeration, precipitation, and/or micellization. PEs enabled the selective recovery of the more hydrophobic DCA 16 and DCA 18 over DCA 12, confirming the dominant role of chain length in LCDA separation. In contrast, RE with Aliquat®336 maximized total LCDA recovery, achieving extraction efficiencies above 85%, but with reduced selectivity. Validation in autoclaved fermentation broth from UCO feedstock confirmed the potential of Aliquat®336 in octanol for high LCDA recovery, while revealing lower extraction efficiencies than in model mixtures due to broth matrix complexity. Overall, this study establishes LLE as a promising platform for LCDA recovery and highlights that future downstream process design must balance total recovery, chain-length selectivity, and broth-specific matrix effects.

Long-chain dicarboxylic acids (LCDAs) are valuable chemicals with a variety of industrial applications. However, since they are typically fossil-derived, there is a need for renewable feedstocks. This study investigated grease trap waste (GTW), an underutilised waste stream, for LCDA production using Candida tropicalis. Several experiments optimised glucose and GTW feed rates and pH, resulting in a final titre of 34.7 g/L LCDAs with a productivity of 0.42 g/(L·h) and a yield of 76 % after 140 h. Optimal conditions included a glucose feed rate of 0.38 g/(L·h), as lower feed rates resulted in glucose deficiency, while higher glucose feed rates promoted excessive growth. The optimal GTW feed rate was 0.5 g/(L·h), as higher rates had inhibitory effects on yeast growth and LCDA production. However, maintaining a pH of 7 was essential to limit foaming, deviating from typical alkaline conditions. This work demonstrated GTW's potential for sustainable LCDA biosynthesis, supporting circular bioeconomy goals.

Grease trap waste (GTW) is a lipid-rich, low-value waste stream that is a promising feedstock for the production of long-chain dicarboxylic acids (LCDAs) by Candida viswanathii. However, the presence of anionic surfactants and trace metals hinders this bioconversion process. This study evaluated the use of ceramic ultrafiltration as a pretreatment technology. A 3 nm ZrO2 ceramic membrane removed approximately 90% of anionic surfactants and over 90% of Ni, Zn and Fe. Using a C1-grafted variant increased the flux sevenfold, to 2.3 L/(m2.h), while retaining the same level of removal. Pretreating the GTW increased LCDA titres in shake flasks by up to sixfold compared to untreated feed. In fed-batch bioreactor runs, pretreated GTW yielded 43.5 g/L LCDA, which is a 25% improvement to the untreated GTW, with a final productivity of 0.46 g/(L.h) and a lipid-to-LCDA yield of 82%. However, biomass formation was lower with pretreated GTW, likely reflecting reduced levels of growth-promoting cofactors. Nevertheless, these results demonstrate that ceramic ultrafiltration enables robust LCDA production from GTW, supporting valorisation of lipid wastes.

Genetic modification of lignocellulosic biomass can enhance its processability by reducing its recalcitrance to pretreatment and enzymatic conversion to chemicals such as biofuels. Downregulation of cinnamyl alcohol dehydrogenase 1 (CAD1) in poplar results in the increased incorporation of sinapaldehyde in the lignin polymer. Here, we investigated steam explosion as a pretreatment strategy for the enzymatic hydrolysis of field-grown CAD1-downregulated and wild-type (WT) poplar. Steam explosion at 210°C for 90 s resulted in a significantly higher lignin reduction of 25% for CAD wood, compared to 15% for WT wood. Under limited conditions at 8.4% solid loading and 8 filter paper units of Cellic Ctec3 HS per gram dry weight, enzymatic hydrolysis resulted in a significantly higher average glucose yield of 76% for CAD wood compared to 60% for WT wood. Our data show that steam explosion pretreatment of CAD1-modified poplar is a promising strategy to improve enzymatic hydrolysis without the need for additional chemicals.

A major obstacle to employing the full potential of lignin-based aromatics is the fractionation of the monomers present in lignin, specifically the separation of guaiacyl (G) and syringyl (S) units, which possess nearly identical molecular weights (196 Da versus 166 Da) and dimensions, and identical functional groups. Such similarities make their separation highly challenging using conventional techniques and are generally considered beyond the capabilities of size-based membrane processes. This study examines the feasibility of organic solvent nano-filtration for fractionation of guaiacyl and syringyl units, and how membrane and process parameters affect separation of these two molecules. Sixteen commercially available membranes were tested with methanol and ethyl acetate as solvents. The results demonstrate that, despite the extreme similarity of the solutes, selective separation is achievable and is primarily governed by membrane material and solvent selection rather than the pore size-based molecular weight cut-off. Polyimide-based solvent-resistant membranes exhibited the highest selectivity, with a maximum observed separation factor of 3.33 obtained using a DuraMemTM 500 membrane in methanol. These findings demonstrate the potential of nanofiltration to address previously unresolved separation challenges in lignin valorization and provide a basis for further process development.

Bio-based long-chain dicarboxylic acids (LCDAs) are in high demand in the polymer industry. These compounds have diverse applications as building blocks for polymers with distinct features, which lead to a fast-growing global LCDA market. However, bio-based LCDA production is currently limited in Europe as established processes are using the pathogenic yeast, Candida tropicalis. Therefore, this study aimed to establish safe and sustainable LCDA production using an industrially relevant non-pathogenic yeast, Starmerella bombicola. The metabolic network was successfully controlled to channel fatty acids from rapeseed oil into the omega-oxidation for the high production of LCDAs. Importantly, the engineered yeast strain produced 5.5 g/l of total LCDAs in shake flasks. Furthermore, pH optimization of the bioprocess resulted in a significant improvement of the total LCDA titer up to 117.8 g/l. The outcomes strongly demonstrate that S. bombicola can serve as a safe and efficient platform microorganism for industrial LCDA production.

Long-chain dicarboxylic acids are versatile aliphatic compounds with two terminal carboxyl groups and hydrocarbon chains of 12 or more carbons, offering tunable thermal, mechanical, and chemical properties. They are widely used in polymers, lubricants, adhesives, pharmaceuticals, and other industrial chemicals. Conventional chemical synthesis relies on fossil feedstocks, harsh reagents, and energy-intensive processes, generating hazardous by-products and raising sustainability concerns. Microbial production has emerged as a greener alternative, exploiting α- and ω-oxidation pathways in oleaginous yeasts such as Candida viswanathii and Yarrowia lipolytica. Metabolic and process engineering—including β-oxidation disruption, cytochrome P450 overexpression, and optimized fed-batch cultivation—has improved titers and productivities. Renewable hydrophobic and hydrophilic feedstocks, including plant oils, lignocellulosic sugars, and industrial wastes, enhance sustainability. Beyond canonical LCDAs, enzymatic modifications enable the production of functionalized derivatives, including hydroxy fatty acids, α,ω-diols, α,ω-diamines, and amino carboxylic acids, broadening their applications in bio-based polymers. This mini review provides an overview of long-chain dicarboxylic acid production, covering chemical synthesis routes and microbial approaches. It focuses on microbial production strains, feedstock strategies, metabolic and process engineering, and the biosynthesis of LCDA derivatives as integrated approaches toward sustainable and industrially viable LCDA biomanufacturing.

Valorization of lignocellulosic biomass for sustainable production of high-value chemicals is challenged by the complexity of lignin, a phenolic biopolymer. Beyond the classical lignin monomers derived from p-coumaryl, coniferyl, and sinapyl alcohol, grass lignins incorporate substantial amounts of monolignol p-coumarates that are produced by p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE (PMT). Here, the CRISPR/Cas9-mediated mutation of ZmPMT1 in maize enabled the design of biomass depleted in p-coumaroylated lignin and enriched in guaiacyl lignin. Lignin-first biorefining of stem biomass from zmpmt1 mutants by reductive catalytic fractionation (RCF) generated a lignin oil depleted in carboxylates and enriched in guaiacyl-derived alcohols, which are desirable substrates for bio-based polyurethane synthesis. The reported lignin engineering in maize is a promising strategy for designing a dual-purpose crop, providing both food and feed, along with a renewable feedstock for the production of plant-based chemicals.

There is a rapidly growing global market of long-chain dicarboxylic acids (LCDAs) used in polymer manufacturing and other material-related industrial applications. Various types of LCDAs are considered highly value-added building blocks due to their ability to diversify the properties of the end products. Nevertheless, industrial access to very long-chain dicarboxylic acids (VLCDAs, ≥C20) is largely limited, posing a challenge to the sustainable production of green polymers. Therefore, the purpose of this study is to expand the bio-based LCDA repertoire in a non-pathogenic, oleaginous and industrially well-established yeast, Starmerella bombicola to overcome these limitations. To produce VLCDAs, an endogenous Cyp52 enzyme with a strong activity for hydroxylation of very long-chain fatty acids (VLCFAs) was selected and the intracellular fatty acid pool was modified by introducing fatty acid elongases. Furthermore, the fatty acid channeling into the ω-oxidation was strengthened. The combined metabolic engineering strategy significantly improved the production of VLCDAs from 0.3 g/L to 2.3 g/L using rapeseed oil as feedstock. The results establish an efficient bio-based route for VLCDA synthesis in S. bombicola, highlighting its potential to significantly contribute to the supply of diverse and renewable bio-based building blocks for industrial applications.

The outflow of pharmaceutically active chemicals (PhACs) exerts a negative impact on biological systems even at extremely low concentrations. For instance, enormous threats to human and aquatic species have resulted from the widespread use of antibiotics in ecosystems, which stimulate the emergence and formation of antibiotic-resistant bacterial species and associated genes. Additionally, it is challenging to eliminate these PhACs by employing conventional physicochemical water treatment techniques. Enzymatic approaches, including laccase, have been identified as a promising alternative to eliminate a broad array of PhACs from water matrices. However, their application in environmental bioremediation is hindered by several factors, including the enzyme's stability and its location in the aqueous environment. Such obstacles may be surmounted by employing laccase immobilization, which enables enhanced stability (including inactivation caused by the substrate), and thus improved catalysis. This review emphasizes the potential hazards of PhACs to aquatic organisms within the detection concentration range of ngL-1 to µgL-1, as well as the deployment of laccase-based multifunctional biocatalytic systems for the environmentally friendly mitigation of anticancer drugs, analgesics/NSAIDs, antibiotics, antiepileptic agents, and beta blockers as micropollutants. This approach could reduce the underlying toxicological consequences. In addition, current developments, potential applications, and viewpoints have focused on computer-assisted investigations of laccase-PhACs binding at enzyme cavities and degradability prediction.

Mannosylerythritol lipids (MELs) are a promising group of biosurfactants due to their high fermentation yield, selfassembly and biological activity. During fermentation by Pseudozyma aphidis, a mixture of MELs with different levels of acylation is formed, of which the fully deacetylated form is the most valuable. In order to reduce the environmental impact of deacetylation, an enzymatic process using natural deep eutectic solvents (NADES) has been developed. We tested the deacetylation of a purified MELs mixture with immobilized Candida antarctica lipase B enzyme and 2-ethylhexanol as co-substrate in 140 h reactions with different NADES. We identified hydrophobic NADES systems with similar yields and kinetics as in pure 2-ethylhexanol solvent. Our results indicate that deacetylation of MELs mixtures in NADES as a solvent is possible with yields comparable to pure co-substrate and that hydrophobic NADES without carboxylic acid compounds facilitate the reaction to the greatest extent. Enzymatic deacetylation of mannosylerythritol lipid (MEL) biosurfactant mixtures in naturally derived deep eutectic solvents has comparable yields and kinetics to conventional organic solvents. Here, the example of thymol-coumarin 1 : 1 mixture is shown where the diacetylated (MEL-A) and monoacetylated (MEL-B and MEL-C) are converted to fully deacetylated compound (MEL-D) with 50 % yield after 140 hours.+image

Rhodotorula kratochvilovae has shown to be a promising species for microbial oil production from lignin-derived compounds. Yet, information on R. kratochvilovae's detoxification and microbial oil production is scarce. This study investigated the growth and microbial oil production on the phenolic-containing effluent from poplar steam explosion and its detoxification with five R. kratochvilovae strains (EXF11626, EXF9590, EXF7516, EXF3697, EXF3471) and compared them with Cutaneotrichosporon oleaginosum. The R. kratochvilovae strains reached a maximum growth rate up to four times higher than C. oleaginosum. Furthermore, all R. kratochvilovae strains generally degraded phenolics more rapidly and to a larger extent than C. oleaginosum. However, the diluted substrate limited the lipid production by all strains as the maximum lipid content and titre were 10.5% CDW and 0.40 g/L, respectively.

Lignin removal plays a crucial role in the efficient bioconversion of lignocellulose to fermentable sugars. As a delignification process, fungal pretreatment has gained great interest due to its environmental friendliness and low energy consumption. In our previous study, a positive linear correlation between acid-insoluble lignin degradation and the achievable enzymatic saccharification yield has been found, hereby highlighting the importance of the close follow-up of lignin degradation during the solid-state fungal pretreatment process. However, the standard quantification of lignin, which relies on the two-step acid hydrolysis of the biomass, is highly laborious and time-consuming. Vibrational spectroscopy has been proven as a fast and easy alternative; however, it has not been extensively researched on lignocellulose subjected to solid-state fungal pretreatment. Therefore, the present study examined the suitability of near-infrared spectroscopy (NIR) for the rapid and easy assessment of lignin content in poplar wood pretreated with Phanerochaete chrysosporium. Furthermore, the predictive power of the obtained calibration model and the recently published ATR-FTIR spectroscopy-based model were compared for the first time using the same fungus-treated wood data set. PLSR was used to correlate the NIR spectra to the acid-insoluble lignin contents (19.9%-27.1%) of pretreated wood. After normalization and second derivation, a PLSR model with a good coefficient of determination (RCV2 = 0.89) and a low root mean square error (RMSECV = 0.55%) were obtained despite the heterogeneous nature of the fungal solid-state fermentation. The performance of this PLSR model was comparably good to the one obtained by ATR-FTIR (RCV2 = 0.87) while it required more extensive spectral pre-processing. In conclusion, both methods will be highly useful for the high-throughput and user-friendly monitoring of lignin degradation in a solid-state fungal pretreatment-based biorefinery concept.

Natural deep eutectic solvents (NADES) represent a green alternative to conventional organic solvents as reaction medium, offering more benign properties. To efficiently design NADES for biocatalysis, a better understanding of their effect on these reactions is needed. We hypothesize that this effect can be described by separately considering (1) the solvent interactions with the substrates, (2) the solvent viscosities and (3) the enzyme stability in NADES. We investigated the effect of substrate solvation and viscosity on the reaction rate; and the stability of the enzyme in NADES. To this end, we monitored the conversion over time of the transesterification of vinyl laurate with 1-butanol by the lipase enzyme Candida antarctica B in NADES of different compounds and molar ratios. The initial reaction rate is higher in most NADES (varying between 1.14 and 15.07 μ mol min^-1 mg^-1 ) than in the reference n-hexane (4.0 μ mol min^-1 mg^-1 )), but no clear relationship between viscosity and initial reaction rate was found. The increased reaction rate is most likely related to the solvation of the substrate due to a change in the activation energy of the reaction or a change in the conformation of the substrate. The enzyme retained part of its activity after the first 2 h of reaction (on average 20 μ mol min^-1 mg^-1 ), but this may also be due to slow dissolution of the substrate. The effect of viscosity seems to be marginal next to the effect of solvation and possible enzyme-NADES interaction. The enzyme retains some of its activity during the 24-hour measurements, but the enzyme incubation experiments did not yield accurate, comparable values.

Lignin determination in lignocellulose with the conventional two-step acid hydrolysis method is highly laborious and time-consuming. However, its quantification is crucial to monitor fungal pretreatment of wood, as the increase of acid-insoluble lignin (AIL) degradation linearly correlates with the achievable enzymatic saccharification yield. Therefore, in this study, a new attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy method was developed to track fungal delignification in an easy and rapid manner. Partial least square regression (PLSR) with cross-validation (CV) was applied to correlate the ATR-FTIR spectra with the AIL content (19.9 %-27.1 %). After variable selection and normalization, a PLSR model with a high coefficient of determination (RCV2 = 0.87) and a low root mean square (RMSECV = 0.60 %) were obtained despite the heterogeneous nature of the fungal solid-state fermentation. These results show that ATR-FTIR can reliably predict the AIL content in fungus-treated wood while being a high-throughput method. This novel method can facilitate the transition to the wood-based economy.

Pretreatment is crucial for the conversion of lignocellulose to biofuels. Unlike conventional chemical/physicochemical methods, fungal pretreatment uses white-rot fungi and mild reaction conditions. However, challenges, including substrate sterilization, long duration, and low sugar yields associated with this method, contribute to lower techno-economic performance, an aspect that has rarely been investigated. This study aimed to evaluate the feasibility of fungal pretreatment of nonsterilized poplar wood. Various factors, including inoculum types, fermentation supplements, and cultivation methods, were investigated to optimize the process. A techno-economic assessment of the optimized processes was performed at a full biorefinery scale. The scenario using nonsterilized wood as a substrate, precolonized wood as an inoculum, and a 4 week pretreatment showed a 14.5% reduction in sugar production costs (2.15/kg) compared to using sterilized wood. Although the evaluation of nonsterilized wood pretreatment showed promising cost reductions, fungal pretreatment remained more expensive than conventional methods due to the significant capital investment required.

The production of chemicals through a circular economy is becoming increasingly important and one way to achieve this is through the utilisation of waste streams. One such waste stream that holds potential for production of chemicals through biochemical methods is grease trap waste (GTW). However, before this waste stream can be used in bioprocesses, it is crucial to understand its effects on the process. This study explores the toxicity of the GTW on the growth of Candida tropicalis ATCC 20962, a yeast that is applied to convert lipids into valuable long -chain dicarboxylic acids (LCDAs). The GTW is characterised and the inhibition of the different components on the growth of C. tropicalis is examined. Results indicate significant inhibition of yeast growth at a concentration of 40 g/L of GTW and higher. Further analysis confirms that the presence of anionic detergents may partially contribute to this inhibition. In contrast, the pesticides, free fatty acids and heavy metals did not appear to significantly affect the growth of C. tropicalis. Although this study suggests that GTW could be a suitable feed-stock for Candida tropicalis ATCC 20962, more research is required to identify other potentially toxic components and to determine if pre-treatment is necessary for industrial applications.

During the conversion of lignocellulose, phenolic wastewaters are generated. Therefore, researchers have investigated wastewater valorization processes in which these pollutants are converted to chemicals, i.e., lipids. However, wastewaters are lean feedstocks, so these valorization processes in research typically require the addition of large quantities of sugars and sterilization, which increase costs. This paper investigates a repeated batch fermentation strategy with Rhodotorula kratochvilovae EXF7516 and Cutaneotrichosporon oleaginosum ATCC 20509, without these requirements. The pollutant removal and its conversion to microbial oil were evaluated. Because of the presence of non-monomeric substrates, the ligninolytic enzyme activity was also investigated. The repeated batch fermentation strategy was successful, as more lipids accumulated every cycle, up to a total of 5.4 g/L (23% cell dry weight). In addition, the yeasts consumed up to 87% of monomeric substrates, i.e., sugars, aromatics, and organics acids, and up to 23% of non-monomeric substrates, i.e., partially degraded xylan, lignin, cellulose. Interestingly, lipid production was only observed during the harvest phase of each cycle, as the cells experienced stress, possibly due to oxygen limitation. This work presents the first results on the feasibility of valorizing non-sterilized lignocellulosic wastewater with R. kratochvilovae and C. oleaginosum using a cost-effective repeated batch strategy.

Natural deep eutectic solvents (NADES) represent a green alternative to common organic solvents in the biochemical industry due to their benign behavior and tailorable properties, in particular as media for enzymatic reactions. However, to fully exploit their potential in enzymatic reactions, there is a need for a more fundamental understanding of how these neoteric solvents influence the course of these reac-tions. Thus, the aim of this study is to investigate the influence of NADES with various molar composi-tions on the stability and structure of enzymes, applying molecular dynamics simulations. This can help to better understand the effect of individual compounds of NADES, in addition to eutectic mixtures. More specifically, we simulate the behavior of Candida antarctica lipase B (CALB) enzyme in NADES com-posed of choline chloride with either urea, ethylene glycol or glycerol. Hereto, we monitor the NADES microstructure, the general stability of the enzyme and changes in the structure of its active sites and sur-face residues. Our simulations show that none of the studied NADES systems significantly disrupt the microstructure of the solvent or the stability of the CALB enzyme within the time scales of the simula-tions. The enzyme preserves its initial structure, size and intra-chain hydrogen bonds in all investigated compositions and, for the first time reported, also in NADES with increased hydrogen bond donating com-pound ratios. As the main novelty, our results indicate that, in addition to the composition, the molar ratio can be an additional variable to fine-tune the physicochemical properties of NADES without altering the enzyme characteristics. These findings could facilitate the development and application of task -tailored NADES media for biocatalytic processes. (c) 2022 Elsevier B.V. All rights reserved.

Waste paper is recovered and bleached to produce recycled newsprints and magazines. It is composed of a fibre mixture from different wood pulping processes. Each type of fibre shows a different reactivity towards bleaching. Consequently, if the composition of waste paper changes over time, the actual industrial bleaching process may no longer be suitable to achieve the intended brightness. This study aims to develop a multiple linear regression that correlates brightness and fibre composition to determine in advance whether a waste paper stream can achieve the intended brightness. Several samples of four of the most representative fibre types were bleached under specific laboratory conditions, and the resulting brightness was used to develop the regression. The resulting model is valid and consistent when the amount of bleached fibre chemically pulped type in the mixed fibre stream does not exceed 80%. Waste samples with a known fibre composition were then bleached to verify the model. The measured brightness followed the same trend predicted by the regression but was lower at a constant value. The use of a correction factor allowed for a good fit. The cause of this discrepancy could be the differences between the reference fibre mixtures and waste paper pulp not included in the model (e.g. contaminants or collapsed fibres). This work is a first step to develop a simple statistical tool to estimate the brightness of waste paper pulp, despite some limitations.