Unleashing the capacity of Rhodococcus for converting lignin into lipids.

Bioconversion of bioresources/wastes (e.g., lignin, chemical pulping byproducts) represents a promising approach for developing a bioeconomy to help address growing energy and materials demands. Rhodococcus, a promising microbial strain, utilizes numerous carbon sources to produce lipids, which are precursors for synthesizing biodiesel and aviation fuels. However, compared to chemical conversion, bioconversion involves living cells, which is a more complex system that needs further understanding and upgrading. Various wastes amenable to bioconversion are reviewed herein to highlight the potential of Rhodococci for producing lipid-derived bioproducts. In light of the abundant availability of these substrates, Rhodococcus' metabolic pathways converting them to lipids are analyzed from a "beginning-to-end" view. Based on an in-depth understanding of microbial metabolic routes, genetic modifications of Rhodococcus by employing emerging tools (e.g., multiplex genome editing, biosensors, and genome-scale metabolic models) are presented for promoting the bioconversion. Co-solvent enhanced lignocellulose fractionation (CELF) strategy facilitates the generation of a lignin-derived aromatic stream suitable for the Rhodococcus' utilization. Novel alkali sterilization (AS) and elimination of thermal sterilization (ETS) approaches can significantly enhance the bioaccessibility of lignin and its derived aromatics in aqueous fermentation media, which promotes lipid titer significantly. In order to achieve value-added utilization of lignin, biodiesel and aviation fuel synthesis from lignin and lipids are further discussed. The possible directions for unleashing the capacity of Rhodococcus through synergistically modifying microbial strains, substrates, and fermentation processes are proposed toward a sustainable biological lignin valorization.

Ethylenediamine pretreatment simultaneously improved carbohydrate hydrolysis and lignin valorization.

Lignocellulosic biomass (LCB) is the promising feedstock for value-added products, which would contribute to the bioeconomy and sustainable development. The efficient pretreatment is still required in the biorefinery of LCB. To make a simultaneous utilization of carbohydrates and lignin, a novel easy-recycled ethylenediamine (EDA) pretreatment was designed and evaluated in the present study. The results highlighted that this pretreatment yielded 96% glucose and 70% xylose in enzymatic hydrolysis. It simultaneously promoted the depolymerization of lignin into small molecules and functionalized the yielded lignin with Schiff base and amide structures. These animated-lignins showed a pH-responsive behavior and the excellent flocculation capacity by reducing more than 90% turbidity of kaolin suspensions. Therefore, easy-recycled EDA pretreatment hold the promise to simultaneously enhance the enzymatic hydrolysis of carbohydrates and endowed the new functionality of lignin toward downstream valorization, which improved the process feasibility and potentially enable the sustainability of LCB utilization.

Steam explosion pretreatment coupling high-temperature short-time sterilization facilitating cellulose degradation and sporulation-regulatory gene expression in high-solid fermentation.

Steam explosion coupling high-temperature short-time sterilization (SE-HTST) was exploited to modify cellulosic biomass medium properties and promote high-solid fermentation (HSF). Biomass characterization analysis showed that SE-HTST enlarged microstructural pores and cavities in solid media, providing more effective space for microbial growth. Meanwhile, SE-HTST helped to release glucose from the cellulose with 35.8 ± 4.5, 20.0 ± 2.3, and 12.3 ± 5.7 mg glucose/g dry medium at 24, 48, and 72 h of fermentation, which were 3.1, 2.3, and 1.5 times higher than that in medium from conventional thermal sterilization (CTS), respectively. SE-HTST increased the viable cell and spore number of Bacillus subtilis by 1.8 and 1.6 times at 72 h of fermentation compared to CTS. Moreover, the expressions of master transcriptional gene spo0A and the early sigma factors of sigF and sigE genes gradually increased in the SE-HTST medium, showing enhanced sporulation in HSF. Therefore, SE-HTST is an effective strategy for facilitating cellulose degradation, improving glucose nutrients in biomass medium, and promoting sporulation-regulatory gene expression during high-solid fermentation, which enhances the production of microbial ecological agents using B. subtilis significantly.

Surfactant-assisted ethylenediamine for the deconstruction and conversion of corn stover biomass.

Lignocellulosic biomass is a promising feedstock to produce sustainable fuels and energy toward a green bioeconomy. A surfactant-assisted ethylenediamine (EDA) was developed for the deconstruction and conversion of corn stover in this study. The effects of surfactants on the whole conversion process of corn stover was also evaluated. The results showed that xylan recovery and lignin removal in solid fraction were significantly enhanced by surfactant-assisted EDA. The glucan and xylan recoveries in solid fraction reached 92.1% and 65.7%, respectively, while the lignin removal was 74.5% by sodium dodecyl sulfate (SDS)-assisted EDA. SDS-assisted EDA also improved the sugar conversion in 12 h enzymatic hydrolysis at low enzyme loadings. The ethanol production and glucose consumption of washed EDA pretreated corn stover in simultaneous saccharification and co-fermentation were improved with the addition of 0.001 g/mL SDS. Therefore, surfactant-assisted EDA showed the potential to improve the bioconversion performance of biomass.

Biological valorization of lignin to flavonoids.

Lignin is the most affluent natural aromatic biopolymer on the earth, which is the promising renewable source for valuable products to promote the sustainability of biorefinery. Flavonoids are a class of plant polyphenolic secondary metabolites containing the benzene ring structure with various biological activities, which are largely applied in health food, pharmaceutical, and medical fields. Due to the aromatic similarity, microbial conversion of lignin derived aromatics to flavonoids could facilitate flavonoid biosynthesis and promote the lignin valorization. This review thereby prospects a novel valorization route of lignin to high-value natural products and demonstrates the potential advantages of microbial bioconversion of lignin to flavonoids. The biodegradation of lignin polymers is summarized to identify aromatic monomers as momentous precursors for flavonoid synthesis. The biosynthesis pathways of flavonoids in both plants and strains are introduced and compared. After that, the key branch points and important intermediates are clearly discussed in the biosynthesis pathways of flavonoids. Moreover, the most significant enzyme reactions including Claisen condensation, cyclization and hydroxylation are demonstrated in the biosynthesis pathways of flavonoids. Finally, current challenges and potential future strategies are also discussed for transforming lignin into various flavonoids. The holistic microbial conversion routes of lignin to flavonoids could make a sustainable production of flavonoids and improve the feasibility of lignin valorization.

Creative biological lignin conversion routes toward lignin valorization.

Lignin, the largest renewable aromatic resource, is a promising alternative feedstock for the sustainable production of various chemicals, fuels, and materials. Despite this potential, lignin is characterized by heterogeneous and macromolecular structures that must be addressed. In this review, we present biological lignin conversion routes (BLCRs) that offer opportunities for overcoming these challenges, making lignin valorization feasible. Funneling heterogeneous aromatics via a 'biological funnel' offers a high-specificity bioconversion route for aromatic platform chemicals. The inherent aromaticity of lignin drives atom-economic functionalization routes toward aromatic natural product generation. By harnessing the ligninolytic capacities of specific microbial systems, powerful aromatic ring-opening routes can be developed to generate various value-added products. Thus, BLCRs hold the promise to make lignin valorization feasible and enable a lignocellulose-based bioeconomy.

Bacterial conversion routes for lignin valorization.

As the largest renewable aromatic resource, lignin is a promising feedstock for production of value-added products. However, lignin valorization has not been implemented due to the recalcitrant and heterogeneity of lignin. Herein, this work provides a systematic overview of bacterial lignin valorization for producing value-added products from the viewpoint of a cascaded conversion route. The combinatorial depolymerization strategy facilitates the yield of a lignin-derived aromatic stream suitable for the bacterial conversion. Bacterial active transports are curial to improve the uptake of lignin-derived aromatics. Intracellular metabolic pathways of bacteria assimilate heterogenous lignin-derived aromatics through "biological funnel" into central aromatic intermediates. These intermediates can be effectively metabolized in bacteria through aromatic ring cleavage pathways to enable the biosynthesis of various value-added products. The techno-economic analysis highlights that bacterial conversion improves the feasibility of co-production of value-added products from lignin. Therefore, the bacterial cascaded conversion routes hold great promise for upgrading heterogeneous lignin into value-added products and thus contribute to the profitability of lignin valorization.

Identifying ligninolytic bacteria for lignin valorization to bioplastics.

Biological valorization of lignin to bioplastics is a promising route to improve biorefinery efficiency and address environmental challenges. A two-stage screening procedure had been designed to successfully identify four ligninolytic bacteria from soil samples. The isolated bacteria displayed substrate preference of guaiacyl- and hydroxyphenyl-based aromatics, but they effectively synthesized polyhydroxyalkanoates (PHAs). B. cepacia B1-2 and P. putida KT3-1 accumulated 27.3% and 20.9% PHA in cells and achieved a titer of 280.9 and 204.1 mg/L, respectively, from p-hydroxybenzoic acid. The isolated bacteria exhibited good ligninolytic performance indicated by the degradation of ß-O-4 linkage and small molecules. B. cepacia B1-2 grew well on actual lignin substrate and yielded a PHA titer of 87.2 mg/L. With the design of fed-batch mode, B. cepacia B1-2 produced the highest PHA titer of 1420 mg/L from lignin-derived aromatics. Overall, isolated ligninolytic bacteria show good PHA accumulation capacity, which are the promising host strains for lignin valorization.

Emerging Modification Technologies of Lignin-based Activated Carbon toward Advanced Applications.

Lignin-based activated carbon (LAC) is a promising high-quality functional material due to high surface area, abundant porous structure, and various functional groups. Modification is the most important step to functionalize LAC by altering its porous and chemical properties. This Review summarizes the state-of-the-art modification technologies of LAC toward advanced applications. Promising modification approaches are reviewed to display their effects on the preparation of LAC. The multiscale changes in the porosity and the surface chemistry of LAC are fully discussed. Advanced applications are then introduced to show the potential of LAC for supercapacitor electrode, catalyst support, hydrogen storage, and carbon dioxide capture. Finally, the mechanistic structure-function relationships of LAC are elaborated. These results highlight that modification technologies play a special role in altering the properties and defining the functionalities of LAC, which could be a promising porous carbon material toward industrial applications.

Cosolvent enhanced lignocellulosic fractionation tailoring lignin chemistry and enhancing lignin bioconversion.

Cosolvent Enhanced Lignocellulosic Fractionation (CELF) is an emerging solvolysis pretreatment to fractionate lignocellulosic biomass. Herein, the bioconversion performance of CELF lignin was fully evaluated for the first time. Results showed that CELF lignin possessed higher content of carboxylic acid OH, lower molecular weight, and disappeared ß-O-4 and ß-5 linkages compared to other two technical lignins including a conventional ethanol organosolv lignin (EOL) and a kraft lignin (KL). Rhodococcus opacus PD630 cell count from CELF lignin fermentation reached the highest value of 3.9 × 107 CFU/mL, representing a 62.5% and 77.3% improvement over EOL and KL, respectively. Correspondingly, lipid yield reached 143 mg/L from CELF lignin, which was 36.2% and 26.5% higher than from EOL and KL, respectively. Principal component analysis (PCA) revealed that more carboxylic acid groups and lower molecular weight contributed to the enhanced bioconversion performance of CELF lignin. This study demonstrates that CELF lignin is a promising candidate for bioconversion.

Transforming biorefinery designs with 'Plug-In Processes of Lignin' to enable economic waste valorization.

Biological lignin valorization has emerged as a major solution for sustainable and cost-effective biorefineries. However, current biorefineries yield lignin with inadequate fractionation for bioconversion, yet substantial changes of these biorefinery designs to focus on lignin could jeopardize carbohydrate efficiency and increase capital costs. We resolve the dilemma by designing 'plug-in processes of lignin' with the integration of leading pretreatment technologies. Substantial improvement of lignin bioconversion and synergistic enhancement of carbohydrate processing are achieved by solubilizing lignin via lowering molecular weight and increasing hydrophilic groups, addressing the dilemma of lignin- or carbohydrate-first scenarios. The plug-in processes of lignin could enable minimum polyhydroxyalkanoate selling price at as low as $6.18/kg. The results highlight the potential to achieve commercial production of polyhydroxyalkanoates as a co-product of cellulosic ethanol. Here, we show that the plug-in processes of lignin could transform biorefinery design toward sustainability by promoting carbon efficiency and optimizing the total capital cost.

Emerging Strategies for Modifying Lignin Chemistry to Enhance Biological Lignin Valorization.

Biological lignin valorization represents a promising approach contributing to sustainable and economic biorefineries. The low level of valuable lignin-derived products remains a major challenge hindering the implementation of microbial lignin conversion. Lignin's properties play a significant role in determining the efficiency of lignin bioconversion. To date, despite significant progress in the development of biomass pretreatment, lignin fractionation, and fermentation over the last few decades, little efforts have gone into identifying the ideal lignin substrates for an efficient microbial metabolism. In this Minireview, emerging and state-of-the-art strategies for biomass pretreatment and lignin fractionation are summarized to elaborate their roles in modifying lignin structure for bioconversion. Fermentation strategies aimed at enhancing lignin depolymerization for microbial utilization are systematically reviewed as well. With an improved understanding of the ideal lignin structure elucidated by comprehensive metabolic pathways and/or big data analysis, modifying lignin chemistry could be more directional and effective. Ultimately, together with the progress of fermentation process optimization, biological lignin valorization will become more competitive in biorefineries.

Mechanical property of different corn stover morphological fractions and its correlations with high solids enzymatic hydrolysis by periodic peristalsis.

Selective structure fractionation combined with periodic peristalsis was exploited to improve the conversion performance of corn stover. The increase of glucan and lignin content and the decrease of xylan content in stem pith were highest after SE, whereas they were lowest in stem node. Glucan conversion increased in this order: steam node

Xylose production from corn stover biomass by steam explosion combined with enzymatic digestibility.

A novel conversion process using steam explosion combined with enzymatic digestibility was exploited to increase sugar yield. Results showed that glucan and xylan recovery decreased with the increase of holding temperature and residence time in SE, respectively, while glucan and xylan conversion exhibited an opposite trend. The optimal conditions of steam explosion were 160 °C and 48 min, under which glucan and xylan recovery was 93.4% and 71.6%, respectively. Glucan and xylan conversion at 18% solid loading by periodic peristalsis increased by 3.4-5.8% and 4.5-6.2%, respectively, compared with that by water baths shaker. In the whole process, glucose, xylose and total sugar yield reached to 77.3%, 62.8% and 72.3%, respectively. The yield of hydroxymethyl furfural, furfural and lignin-derived products was 6.3 × 10(-2), 7.5 × 10(-2) and less than 3.7 × 10(-2) g/100 g feedstock, respectively. This novel conversion process increased sugar recovery, reduced degradation products formation, improved digestibility efficiency, and hence increased sugar yield.

High temperature aqueous ammonia pretreatment and post-washing enhance the high solids enzymatic hydrolysis of corn stover.

Aqueous ammonia pretreatment was optimized and the limiting factors in high solids enzymatic hydrolysis were assessed. The recommended pretreatment condition to achieve high enzymatic yield was: 180 °C, 20% (w/w) ammonia, 30 min, and 20% solids content. FT-IR and GC-MS results indicated that most of the lignin was degraded to soluble fragments after pretreatment. The pretreated solids after post-washing showed higher enzymatic digestibility at high solids loading than that without washing. The washed solids required lower cellulase and xylanase dosage than unwashed solids to achieve high sugar yield. Enzymatic conversions were declined with the increased solids loading of pretreated solids, pretreated-washed solids, and filter papers. The results indicated that solids loading in enzymatic hydrolysis was an important factor affecting sugar yield. The increasing concentration of glucose and ligno-phenolics mainly inhibited the enzymatic hydrolysis of aqueous ammonia pretreated corn stover.

Evaluation of storage methods for the conversion of corn stover biomass to sugars based on steam explosion pretreatment.

Effects of dry and wet storage methods without or with shredding on the conversion of corn stover biomass were investigated using steam explosion pretreatment and enzymatic hydrolysis. Sugar conversions and yields for wet stored biomass were obviously higher than those for dry stored biomass. Shredding reduced sugar conversions compared with non-shredding, but increased sugar yields. Glucan conversion and glucose yield for non-shredded wet stored biomass reached 91.5% and 87.6% after 3-month storage, respectively. Data of micro-structure and crystallinity of biomass indicated that corn stover biomass maintained the flexible and porous structure after wet storage, and hence led to the high permeability of corn stover biomass and the high efficiency of pretreatment and hydrolysis. Therefore, the wet storage methods would be desirable for the conversion of corn stover biomass to fermentable sugars based on steam explosion pretreatment and enzymatic hydrolysis.

Mass balance and transformation of corn stover by pretreatment with different dilute organic acids.

Previous studies indicated high xylose yield could be achieved after pretreatment using organic acids, but it is necessary to systematically investigate the effects of different parameters during organic acid pretreatments. Corn stover was pretreated with sulfuric, oxalic, citric, tartaric and acetic acid at 50 and 90 mM from 130 to 190°C. The xylan balance for each different acid was distinct, but all balances were very close to 100% by determining xylan recovery, xylooligomer yield, xylose yield and furfural yield. The effects of combined severity on the recovery or yields of these components were also studied. The acid pK(a) value affected the proportion of xylan degradation products. The maximum value of xylose and xylooligomer yield for specific acid pretreatment was also determined by pK(a) value. The maximum xylose yield was obtained after pretreatment with sulfuric and oxalic acid, but more xylooligomers were obtained after pretreatment with weaker acids.