Hydrolysis Of Residues Research Articles

Solubilization and structural changes of lignin in naked oat stems during subcritical water autohydrolysis

In this study, the solubilization and structural changes of lignin in naked oat stems were investigated under subcritical water autohydrolysis systems (170–210 °C, 0.68–1.85 MPa). In this system, Hemicellulose was preferentially hydrolyzed in the liquid water at elevated temperatures, leading to the production of acetic acid and glucuronic acid, which acidified the reaction system. Under acidic and high-temperature conditions, lignin primarily underwent degradation and condensation reactions. At autohydrolysis temperatures below 190 °C and autohydrolysis pressures below 1.22 MPa, lignin degradation was predominant, realizing a maximum lignin removal of 47.8 % and breakage of numerous β-O-4 bonds from lignin. At autohydrolysis temperatures above 190 °C and autohydrolysis pressures above 1.22 MPa, lignin condensation dominated, with an increase in the amount of organic acids generated upon hemicellulose degradation, leading to condensation reactions with the degraded low-molecular-weight lignin. The degree of lignin condensation was positively correlated with the temperature of the reaction system. This study provides essential insights into the dynamic changes in the structure of lignin in both the hydrolysis residue and hydrolysis solution during subcritical water autohydrolysis.

Production of essential oils and sugar-rich hydrolysate from betel leaves (Piper betle)

The study was carried out to fully utilize betel leaves for the extraction of essential oils and production of sugar-rich hydrolysates from the betel leaves residues. Essential oils in the betel leaves were extracted by hydrodistillation and the betel leaves residues were enzymatically hydrolyzed to obtain sugar-rich hydrolysates. Antioxidant and antibacterial activity of the essential oils and the hydrolysates were investigated. Chemical composition analysis of the betel leaves showed that they contained 2.23% reducing sugars, 21.10% polysaccharides, 68.01 mg/g phenolics, 6.17 mg/g flavonoids, 12.05% ash, and 1.63% tannins. Betel essential oils content was 3.14%, with the main components being eugenol (50.37%), γ-muurolene (9.65%), and α-copaene (8.22%). Betel essential oils exhibited antioxidant activity with the IC50 of 0.13 mg/mL and antibacterial capacity against three strains of bacteria, including Escherichia coli, Samonella sp. and Bacillus cereus. The enzymatic hydrolysis of betel leaves residues using Ultraflo Max with a ratio of enzyme to substrate of 5% for 96 h produced the highest amount of reducing sugars of 10.66 g/L containing 48.31% glucose. The results suggest that betel leaves residues hydrolysate can be used as carbon sources for fermentation processes to produce value-added commodities in further investigation.

Valorization of straw biomass into lignin bio-ink, bacterial cellulose and activated nanocarbon through the trade-off alkali-catalyzed glycerol organosolv biorefinery

Integrated lignocellulosic biorefinery that transforms low-value lignocellulose into multiple value-added products is crucial for a low-carbon sustainable development. Herein, a one-pot alkali-catalyzed glycerol organosolv (ALGO) pretreatment-based integrated biorefinery process to directionally produce lignin bio-inks, bacterial celluloses (BCs), and activated nanocarbons (ANCs) was proposed. ALGO pretreatment could modify the structure of dissolved lignins by glycerolysis. The extracted lignin fragments with high-purity (>90 %), uniform with low molecular weight (2500–4000 of Mw), and abundant hydrophilic aliphatic –OH were promising candidates in 3D-printable inks preparation. The glycerolysis reaction during ALGO process also enabled pretreated substrates toward high hydrolyzability and protected pretreatment liquor from generating fermentable inhibitors, making the solid fractions more liable for enzymatic deconstruction and microbial fermentation. Without any detoxification treatments, hydrolysates from ALGO pretreated substrates were used directly as carbon sources for BCs production, resulting in the BC yields were 56 % higher than that of the commercial glucose. The enzymatic hydrolysis residue and BC fermentation waste liquid were synthesized into ANCs via a facile carbonization process, which could further increase the value of ALGO biorefinery. Calculations showed that this ALGO integrated strategy rendered the conversion of ∼75 % lignins in pretreatment liquor to bio-inks and nearly 100 % solid fractions to BCs and ANCs. Unlike the conventional “lignin-first” route to obtain high-purity cellulose by sacrificing the value-adding conversion ability of by-products, the designed technical route in this work achieved a good trade-off between fractionation ability and subsequent conversion potential, inspiring further exploration of the technical correlation between biomass fractionation and bioproduct processing.

Improved reducing sugars production from enzymatic hydrolysis of rubber wood by surfactant-assisted hydrothermal pretreatment

In this work, a facile and environmentally friendly strategy was proposed for the production of reducing sugars from enzymatic hydrolysis of rubber wood by surfactant-assisted hydrothermal pretreatment. Compared with hydrothermal pretreatment, the addition of surfactants (Tween 40) significantly increased the reducing sugars concentration, the highest reducing sugars concentration was up to 22.47 g/L. After surfactant-assisted pretreatment, the crystallinity index of rubber wood hydrolysis residues increased and then decreased with the addition amount of Tween 40. The surfactant-assisted pretreated rubber wood featured high cellulose and hemicellulose conversion rates of 95.1% and 95.4% respectively, which was 122.2% and 41.1% higher than the hydrothermally pretreated rubber wood. It was also found that surfactant-assisted hydrothermal pretreatment had the same improvement effect on increasing the sugar production of other lignocellulose. The results showed that surfactant-assisted hydrothermal pretreatment is a promising and universal method for developing biomass-based products.

An effective method for the direct crystallization of xylonic acid from fermentation broth of agricultural residue hydrolysate

Xylose is the second most abundant sugar in nature and conversion of xylose to xylonic acid (XA) has become a research hotspot in recent years. Xylonic acid can be applied in the equivalent market niche of gluconic acid, due to their similar physical properties. XA bioproduction and application presents certainly a promising approach for xylose valorization in lignocellulose biorefinery, while the XA crystallization is so far still an unattainable goal. In this paper, an original method was proposed and experimentally investigated for the first time to get XA crystals from fermentation broth. In the XA crystallization, methanol was introduced as buffer-cum-solvent. The H2SO4 was dropwise added into the methanol solution dissolving potassium xylonate that is beforehand produced by fermentation. During H2SO4 blending with methanol, the strong and polar H2SO4 leads to XA diffusion and aggregation. Overall, 2.21 g of xylonic acid crystals was obtained per gram of 98% H2SO4 acidification. NMR detection showed that over 99% purity of XA crystals was finally obtained at the yields of 67.2% from xylonate. The integrated process could provide a practicable and effective technology for the high-qualify XA preparation from xylose derivations and lignocellulose biomass biorefinery.

Characterization of a novel acidophilic, ethanol tolerant and halophilic GH12 β-1,4-endoglucanase from Trichoderma asperellum ND-1 and its synergistic hydrolysis of lignocellulosic biomass

A novel acidophilic GH5 β-1,4-endoglucanase (TaCel12) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 1.5-fold increase). Deglycosylated TaCel12 migrated as a single band (26.5 kDa) in SDS-PAGE. TaCel12 was acidophilic with a pH optimum of 4.0 and displayed great pH stability (>80 % activity over pH 3.0–5.0). TaCel12 exhibited considerable activity towards sodium carboxymethyl cellulose and sodium alginate with Vmax values of 197.97 μmol/min/mg and 119.06 μmol/min/mg, respectively. Moreover, TaCel12 maintained >80 % activity in the presence of 20 % ethanol and 4.28 M NaCl. Additionally, Mn2+, Pb2+ and Cu2+ negatively affected TaCel12 activity, while the presence of 5 mM Co2+ significantly increased the enzyme activity. Analysis of action mode revealed that TaCel12 required at least four glucose (cellotetraose) residues for hydrolysis to yield cellobiose and cellotriose. Site-directed mutagenesis results suggested that Glu133 and Glu217 of TaCel12 are crucial catalytic residues, with Asp116 displaying an auxiliary function. Production of soluble sugars from lignocellulose is a crucial step in bioethanol development, and it is noteworthy that TaCel12 could synergistically yield fermentable sugars from corn stover and bagasse, respectively. Thus TaCel12 with excellent properties will be considered a potential biocatalyst for applications in various industries, especially for lignocellulosic biomass conversion.

Bacterial cellulose/gum Arabic composite production by in-situ modification from lavender residue hydrolysate

In this study, bacterial cellulose/gum Arabic composite (BC/GA) was synthesized by in-situ modification from lavender residue hydrolysate for the first time. The in-situ modification with GA adding showed great beneficial effect for BC/GA synthesis. Both the product (BC or BC/GA) yield and the product (BC or BC/GA) production per sugars consumption increased greatly by the in-situ modification when compared with the fermentation without GA adding (2.90 g/L vs. 0.91 g/L, and 0.461 g/g vs. 0.138 g/g). It is hypothesized that the combination of BC and GA is the main mechanism for the beneficial effect of the in-situ modification, and the scanning electron microscope (SEM) images confirmed this hypothesis. GA adding showed little effect on the rheological properties of lavender residue hydrolysate, and this environment was suitable for the combination of BC and GA. The in-situ modification had an obvious influence on the crystallinity index and the thermal stability of BC/GA, but affected little on its functional groups and cellulose structural framework. Besides BC/GA synthesis and structure, the in-situ modification could also alter the texture properties of BC/GA. Overall, this study can offer some useful information for the biochemical conversion from green and cost-effective lavender residue hydrolysate to attractive biomaterial BC/GA.

Degradation of acrylic-melamine based varnish containing Tinuvin 292 with accelerated exposure in fluorescent UV and condensation

This study explored the growth and fermentation capabilities of four distinct yeast strains, including Saccharomyces cerevisiae var. boulardii CNCM I-745, S. cerevisiae 7012, S. cerevisiae 7028, and S. cerevisiae var. diastaticus BE-134, with the aim of identifying the most suitable strain for the production of a fermented beverage from soybean residue hydrolysate (SRH). The results were as follows: S. cerevisiae 7012 exhibited the most efficient fermentation, with residual sugar content of 2.16 g/100 ml, an ethanol concentration of 1.39% v/v, and a favourable aroma, receiving a sensory score of 4.3/5 points. Within this sensory profile, the aromatic compound 2-phenylethanol was found to be the predominant component. Additionally, S. cerevisiae var. boulardii CNCM I-745 demonstrated superior biomass production ability, achieving a cell density of 7.71 log CFU/ml after 48 hours of fermentation solution also showed the highest antioxidant activity, equivalent to 1.63 mg of ascorbic acid per 100 ml. Consequently, these two yeast strains were selected for combined use in fermentation to leverage the probiotic characteristics of S. cerevisiae var. boulardii CNCM I-745 and the flavouring capacity of S. cerevisiae 7012 in creating fermented beverages from SRH.

Assessment of growth and fermentation of some yeasts on soybean residue hydrolysate

This study explored the growth and fermentation capabilities of four distinct yeast strains, including Saccharomyces cerevisiae var. boulardii CNCM I-745, S. cerevisiae 7012, S. cerevisiae 7028, and S. cerevisiae var. diastaticus BE-134, with the aim of identifying the most suitable strain for the production of a fermented beverage from soybean residue hydrolysate (SRH). The results were as follows: S. cerevisiae 7012 exhibited the most efficient fermentation, with residual sugar content of 2.16 g/100 ml, an ethanol concentration of 1.39% v/v, and a favourable aroma, receiving a sensory score of 4.3/5 points. Within this sensory profile, the aromatic compound 2-phenylethanol was found to be the predominant component. Additionally, S. cerevisiae var. boulardii CNCM I-745 demonstrated superior biomass production ability, achieving a cell density of 7.71 log CFU/ml after 48 hours of fermentation solution also showed the highest antioxidant activity, equivalent to 1.63 mg of ascorbic acid per 100 ml. Consequently, these two yeast strains were selected for combined use in fermentation to leverage the probiotic characteristics of S. cerevisiae var. boulardiiCNCM I-745 and the flavouring capacity of S. cerevisiae 7012 in creating fermented beverages from SRH.

Production of artificial humic acid from corn straw acid hydrolysis residue with biogas slurry impregnation for fertilizer application

This study investigated hydrothermal humification of corn straw acid hydrolysis residue with biogas slurry impregnation, aiming at producing water-soluble artificial humic acid fertilizer for fertilizer application and soil remediation. Hydrothermal humification parameters, including potassium hydroxide concentration (1–3 mol/L), retention time (2–6 h), and temperature (140–180 °C), were investigated using water as the liquid phase. The selected hydrothermal humification condition was 1.5 mol/L potassium hydroxide at 180 °C for 4 h. Moreover, biogas slurry impregnation (0–30 days) was evaluated to improve humic acid yield without introducing additional chemicals or energy input. Biogas slurry as the liquid phase increased the humic acid production by 73.24% with 5 days of impregnation compared to the control due to the alkalinity. The humic acid concentration was sufficient for China's national standard of water-soluble humic acid fertilizers in such conditions. The organic components in biogas slurry were involved in artificial humification as a precursor, forming C–N bonds with humic acid. The product with fortified nitrogen-containing functional groups enhanced the nutrient slow-release characteristics and water retention capabilities. The pot experiment further confirmed that artificial humic acid prepared in this study not only promoted the growth of plants but also achieved soil remediation.

Effect of in-situ biochemical modification on the synthesis, structure, and function of xanthan gum based bacterial cellulose generated from Tieguanyin oolong tea residue hydrolysate

The effect of in-situ biochemical modification on the synthesis, structure, and function of xanthan gum based bacterial cellulose generated from Tieguanyin oolong tea residue hydrolysate was evaluated for the first time. This modification could overcome the inhibitory effect of the hydrolysate and the bacterial cellulose yield with 0.6% xanthan gum addition increased by 260.8% compared with that without xanthan gum addition. Bacterial cellulose and xanthan gum were combined by the in-situ modification and the alteration of fermentation medium rheological properties by xanthan gum addition might be beneficial for their combination. The average diameter of the bacterial cellulose microfibrils was increased by the modification, and it had a great influence on the crystalline structure of the bacterial cellulose. Additionally, both the water absorption and texture properties of the bacterial cellulose was strengthened by the modification. Overall, this modification showed great potential for efficient and effective xanthan gum based bacterial cellulose production.

Fungal Hydrolysis of Food Waste: Review of used Substrates, Conditions, and Microorganisms

Food production generates significant amounts of organic waste that can have negative effects on the environment, such as water, soil and air pollution, due to lack of efficient utilisation solutions and insufficient disposal practices. Fungi have great enzymeproducing abilities that can be used for hydrolysing various types of food waste. Carried out in optimal conditions, fungal hydrolysis of generated food waste can be fast and efficient. Currently, fungal hydrolysis capacity for waste treatment has only been briefly demonstrated in previous studies. This review focuses on and summarizes different practises showing the potential of fungal hydrolysis for the use inefficient resource management. The main accent was put on what organisms and waste have been used in previously conducted studies. In addition, temperature, pH level and glucose recovery yields were reviewed. It was concluded that food waste can be efficiently hydrolysed and used as substrates for downstream production of value-added products. The optimal temperature for fungal hydrolysis is above 45 °C, and optimal pH level can differ depending on the process. The possibility of fungal strain optimization and creation of the enzymeproducing mutant, as well as the use of other GRAS fungi should be investigated. To explore a wider range of waste residues, researchers must collaborate with manufacturers to conduct tests and valorise new residues for fungal hydrolysis.

Utilizing hydrolysis residue from bioethanol production as an additive for solid fuel pellets

Cellulosic bioethanol is produced from the structural carbohydrates (cellulose and hemicellulose) in non-food biomass feedstocks via biochemical conversion. A great amount of non-hydrolyzed material, mainly lignin, is left out as a residual byproduct in this process. For the overall process economy, a possible value added use of this hydrolysis residue is to produce solid fuel pellets to power the bioethanol production itself or residential combustion because lignin has a high net energy value and a low fuel-ash content. Moreover, lignin with its self-bonding behavior can serve as a natural binder if used as an additive to make stronger and more durable pellets for energy and other biorefinery applications. In this study, solid fuel pellets fully or partially made from corn stover hydrolysis residue were evaluated from physical, thermochemical, and hygroscopic perspectives. With 10% hydrolysis residue additive, pellet durability increased from 80.7% to 90.1% and reached 92.8% with further hydrolysis residue addition. Enhancements on pellet mechanical properties were also observed by adding 50% or more hydrolysis residue. Energy density increased from 13.7 GJ/m3 to as high as 17.6 GJ/m3 with the amount of additive increasing from none to 100%. Thermal gravimetric and differential scanning calorimetry results revealed that hydrolysis residue strengthened pellet thermal properties (heating value, thermal stability, and exothermal performance). FTIR and NMR spectra were studied to identify inner-unit linkages in aromatic and side-chain regions of lignin to understand its self-bonding behavior. Pellets also showed increased hydrophobicity and better water resistance when a higher percentage of hydrolysis residue additive was used.

Efficient delignification of wheat straw for microbial lipid production enabled by a novel ternary deep eutectic solvent containing ethylene glycol

Lignocellulose pretreatment often faces the problem of lignin condensation with less attention to biofuel production from total sugars, which significantly hinders biomass fractionation and energy utilization efficiency. The aim of this work was to design a novel ternary deep eutectic solvent (DES) (citric acid: choline chloride: ethylene glycol in a molar ratio of 1:1:2) for pretreatment of wheat straw with a solid/liquid ratio (S/L) of 1:20 (w/w) to achieve non-condensed lignin and high-yield fermentable sugars at 100 °C for 12 h. The introduction of ethylene glycol into ternary DES could capture the formed benzyl carbocation to protect lignin structure, inhibit excessive degradation of hemicellulose but to fermentable sugars (e.g., xylose), and improve the enzymatic digestibility of cellulose (98.73%, w/w). The mechanisms of both lignin fractionation and the in-situ lignin protection during the pretreatment of wheat straw enabled by ternary DES were systematically elucidated. Importantly, Trichosporon cutaneum cultivated in undetoxified DES waste liquid (hemicellulose-rich) and residue hydrolysate (cellulose-rich) could totally afford 8.7 g microbial lipid per 100 g wheat straw, which is superior to previous reports. This green in-situ lignin protection and fractionation strategy provides a new avenue for upgrading lignocellulose to microbial lipids.

Extraction of Eucommia ulmoides gum and microbial lipid from Eucommia ulmoides Oliver leaves by dilute acid hydrolysis.

Eucommia ulmoides gum (EUG) is an important natural biomass rubber material, which is usually extracted from Eucommia ulmoides Oliver (EUO). In the extraction process of EUG, pretreatment is the most important step which can efficiently damage EUG-containing cell wall and improve yield of EUG. The FT-IR, XRD, DSC and TG results showed that the thermal properties and structure of the EUG from the dilute acids hydrolysis residue are similar with that of the EUG directly extracted from EUO leaves (EUGD). EUO leaves hydrolysis with AA had the highest EUG yield (16.1%), which was higher than the EUGD yield (9.5%). In the case of the EUO leaves hydrolysis with 0.33 ~ 0.67 wt% of acetic acid (AA), the total sugar was stable in the range of 26.82-27.67g/L. Furthermore, the EUO leaves acid hydrolysate (AA as reagent) was used as carbon sources for lipid-producing fermentation by Rhodosporidium toruloides. After 120h of fermentation, the biomass, lipid content and lipid yield were 12.13g/L, 30.16% and 3.64g/L, respectively. The fermentation results indicated organic acids were no toxic for Rhodosporidium toruloides and the AA also could be used as carbon source for fermentation.

Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 β-Lactamase.

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25-1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

Optimization of Melanin Production by Streptomyces antibioticus NRRL B-1701 Using Arthrospira (Spirulina) platensis Residues Hydrolysates as Low-Cost L-tyrosine Supplement.

Melanin is a functional pigment that is used in various products. It can be produced by Streptomyces antibioticus NRRL B-1701 when supplemented with L-tyrosine. Arthrospira (Spirulina) platensis is a cyanobacterium with high protein content, including the protein phycocyanin (PC). During PC's extraction, biomass residues are generated, and these residues still contain various amino acids, especially L-tyrosine, which can be used as a low-cost supplement for melanin production. Thus, this study employed a hydrolysate of A. platensis biomass residue for L-tyrosine substitution. The effects of two drying methods, namely, lyophilization and dying via a hot air oven, on the proximate composition and content of L-tyrosine in the biomass residue were evaluated. The highest L-tyrosine (0.268 g L-tyrosine/100 g dried biomass) concentration was obtained from a hot-air-oven-dried biomass residue hydrolysate (HAO-DBRH). The HAO-DBRH was then used as a low-cost L-tyrosine supplement for maximizing melanin production, which was optimized by the response surface methodology (RSM) through central composite design (CCD). Using the RSM-CCD, the maximum level of melanin production achieved was 0.24 g/L, which is approximately four times higher than it was before optimization. This result suggests that A. platensis residue hydrolysate could be an economically feasible and low-cost alternative source of L-tyrosine for the production of melanin.

Combining ABE fermentation and anaerobic digestion to treat with lipid extracted algae for enhanced bioenergy production

As a downstream process output, biobutanol can be produced via acetone, butanol, and ethanol (ABE) fermentation from lipid-extracted algae (LEA), but the leftover residue has not been treated for additional value. In current study, LEA were acid hydrolyzed to extract glucose into the hydrolysate, which was then used for ABE fermentation to produce butanol. In the meantime, anaerobic digestion was performed on the hydrolysis residue to produce methane and release nutrients for algae recultivation. To optimize butanol and methane production, several carbon or nitrogen supplements were applied. The results showed that the hydrolysate produced a high butanol concentration of 8.5 g/L with bean cake supplemented, and the residue co-digested with wastepaper had a higher methane production compared to the direct anaerobic digestion of LEA. The causes of the enhanced performances were discussed. The digestates were reused for algae recultivation and were proved to be effective for algae and oil reproduction. The combined process of ABE fermentation and anaerobic digestion was thus proved a promising technique to treat LEA for economic benefit.

Fermentative production of astaxanthin from sorghum distillery residue by an indigenous Aurantiochytrium sp. CJ6 strain using a continuous-feeding fed-batch process

In this study, Aurantiochytrium sp. CJ6 was cultivated heterotrophically on a waste resource, sorghum distillery residue (SDR) hydrolysate without adding any nitrogen sources. Mild sulfuric acid treatment released sugars that supported the growth of CJ6. Optimal operating parameters (salinity, 2.5%; pH, 7.5; with light exposure) determined using batch cultivation attained biomass concentration and astaxanthin content of 3.72 g/L and 69.32 µg/g dry cell weight (DCW), respectively. Using continuous-feeding fed-batch (CF-FB) fermentation, the biomass concentration of CJ6 increased to 6.3 g/L with biomass productivity and sugar utilization rate of 0.286 mg/L/d and 1.26 g/L/d, respectively. Meanwhile, CJ6 obtained maximum astaxanthin content (93.9 µg/g DCW) and astaxanthin concentration (0.565 mg/L) after 20-day cultivation. Thus, the CF-FB fermentation strategy seems to have a high potential for the cultivation of thraustochytrids to produce the high-value product (astaxanthin) using SDR as the feedstock to achieve circular economy.

X-ray crystallographic and mutational analysis of the NylC precursor: catalytic mechanism of autocleavage and substrate hydrolysis of nylon hydrolase.

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k=2.8 × 10-5 s-1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.