Strategies for high cell density cultivation of Akkermansia muciniphila and its potential metabolism.

IMPORTANCE: Currently, there is significant interest in Akkermansia muciniphila as a promising next-generation probiotic, making it a hot topic in scientific research. However, to achieve efficient industrial production, there is an urgent need to develop an in vitro culture method to achieve high biomass using low-cost carbon sources such as glucose. This study aims to explore the high-density fermentation strategy of A. muciniphila by optimizing the culture process. This study also employs techniques such as LC-MS and RNA-Seq to explain the possible regulatory mechanism of high-density cell growth and increased cell surface hydrophobicity facilitating cell colonization of the gut in vitro culture. Overall, this research sheds light on the potential of A. muciniphila as a probiotic and provides valuable insights for future industrial production.

Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine.

Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.

Computationally Guided Enzymatic Studies on Schizochytrium-Sourced Malonyl-CoA:ACP Transacylase.

The malonyl-CoA:ACP transacylase (MAT) domain is responsible for the selection and incorporation of malonyl building blocks in the biosynthesis of polyunsaturated fatty acids (PUFAs) in eukaryotic microalgae (Schizochytrium) and marine bacteria (Moritella marina, Photobacterium profundum, and Shewanella). Elucidation of the structural basis underlying the substrate specificity and catalytic mechanism of the MAT will help to improve the yield and quality of PUFAs. Here, a methodology guided by molecular dynamics simulations was carried out to identify and mutate specificity-conferring residues within the MAT domain of Schizochytrium. Combining mutagenesis, cell-free protein synthesis, and in vitro biochemical assay, we dissected nearby interactions and molecular mechanisms relevant for binding and catalysis and found that the reorientation of the Ser154 Cß-Oγ bond establishes distinctive proton-transfer chains (His153-Ser154 and Asn235-His153-Ser154) for catalysis. Gln66 can be replaced by tyrosine to shorten the distance between His153 (Nε2) and Ser154 (Oγ), which facilitates a faster proton-transfer rate, allowing better use of acyl substrates than the wild type. Furthermore, we screened a mutant that displayed an 18.4% increase in PUFA accumulation. These findings provide important insights into the study of MAT through protein engineering and will benefit dissecting the molecular mechanisms of other PUFA-related catalytic domains.

Machine learning to predict dynamic changes of pathogenic Vibrio spp. abundance on microplastics in marine environment.

Microplastics are widely found in the marine environment. Recent studies have shown that pathogenic microorganisms can hitchhike on microplastics, which might act as a vector for the spread of pathogens. Vibrio spp. are known to be pathogenic to humans and can cause serious foodborne diseases. In this study, using datasets from an estuary and a mariculture zone in China, five machine learning models were established to predict the relative abundance of Vibrio spp. on microplastics. The results showed that deep neural network (DNN) model and RandomForest algorithm achieved the best predictive performance. Different data sources, data sampling, and processing methods had a little impact on the prediction performance of DNN and RandomForest models. SHapley Additive exPlanations (SHAP) indicated that salinity and temperature are the primary factors affecting the relative abundance of Vibrio spp. The prediction performances of the five machine learning models were further improved by feature selection, providing information to support future experimental research. The results of this study could help establish a long-term and dynamic monitoring system for the relative abundance of Vibrio spp. on microplastics in response to environmental factors as well as provide useful information for assessing the potential health impacts of microplastics on marine ecology and humans.

A robust soft sensor to monitor 1,3-propanediol fermentation process by Clostridium butyricum based on artificial neural network.

With the aggravation of environmental pollution and energy crisis, the sustainable microbial fermentation process of converting glycerol to 1,3-propanediol (1,3-PDO) has become an attractive alternative. However, the difficulty in the online measurement of glycerol and 1,3-PDO creates a barrier to the fermentation process and then leads to the residual glycerol and therefore, its wastage. Thus, in the present study, the four-input artificial neural network (ANN) model was developed successfully to predict the concentration of glycerol, 1,3-PDO, and biomass with high accuracy. Moreover, an ANN model combined with a kinetic model was also successfully developed to simulate the fed-batch fermentation process accurately. Hence, a soft sensor from the ANN model based on NaOH-related parameters has been successfully developed which cannot only be applied in software to solve the difficulty of glycerol and 1,3-PDO online measurement during the industrialization process, but also offer insight and reference for similar fermentation processes.

Development of a fourth-order compact finite difference scheme for simulation of simulated-moving-bed process.

A fourth-order compact finite difference scheme was developed to solve the model equation of simulated moving bed, which has a boundary condition that is updated along the calculation process and cannot be described as an explicit function of time. Two different methods, direct method and pseudo grid point method, were proposed to deal with the boundary condition. The high accuracy of the two methods was confirmed by a case study of solving an advection-diffusion equation with exact solution. The developed compact finite difference scheme was then used to simulate the SMB processes for glucose-fructose separation and enantioseparation of 1,1'-bi-2-naphtol. It was found that the simulated results fit well with the experimental data. Furthermore, the developed method was further combined with the continuous prediction method to shorten the computational time and the results showed that, the computational time can be saved about 45%.

The role of fluconazole in the regulation of fatty acid and unsaponifiable matter biosynthesis in Schizochytrium sp. MYA 1381.

BACKGROUND: Schizochytrium has been widely used in industry for synthesizing polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA). However, unclear biosynthesis pathway of PUFAs inhibits further production of the Schizochytrium. Unsaponifiable matter (UM) from mevalonate pathway is crucial to cell growth and intracellular metabolism in all higher eukaryotes and microalgae. Therefore, regulation of UM biosynthesis in Schizochytrium may have important effects on fatty acids synthesis. Moreover, it is well known that UMs, such as squalene and ß-carotene, are of great commercial value. Thus, regulating UM biosynthesis may also allow for an increased valuation of Schizochytrium. RESULTS: To investigate the correlation of UM biosynthesis with fatty acids accumulation in Schizochytrium, fluconazole was used to block the sterols pathway. The addition of 60 mg/L fluconazole at 48 h increased the total lipids (TLs) at 96 h by 16% without affecting cell growth, which was accompanied by remarkable changes in UMs and NADPH. Cholesterol content was reduced by 8%, and the squalene content improved by 45% at 72 h, which demonstrated fluconazole's role in inhibiting squalene flow to cholesterol. As another typical UM with antioxidant capacity, the ß-carotene production was increased by 53% at 96 h. The increase of squalene and ß-carotene could boost intracellular oxidation resistance to protect fatty acids from oxidation. The NADPH was found to be 33% higher than that of the control at 96 h, which meant that the cells had more reducing power for fatty acid synthesis. Metabolic analysis further confirmed that regulation of sterols was closely related to glucose absorption, pigment biosynthesis and fatty acid production in Schizochytrium. CONCLUSION: This work first reported the effect of UM biosynthesis on fatty acid accumulation in Schizochytrium. The UM was found to affect fatty acid biosynthesis by changing cell membrane function, intracellular antioxidation and reducing power. We believe that this work provides valuable insights in improving PUFA and other valuable matters in microalgae.

Overproduction of L-tryptophan via simultaneous feed of glucose and anthranilic acid from recombinant Escherichia coli W3110: Kinetic modeling and process scale-up.

L-tryptophan is an essential amino acid widely used in food and pharmaceutical industries. However, its production via Escherichia coli fermentation suffers severely from both low glucose conversion efficiency and acetic acid inhibition, and to date effective process control methods have rarely been explored to facilitate its industrial scale production. To resolve these challenges, in the current research an engineered strain of E. coli was used to overproduce L-tryptophan. To achieve this, a novel dynamic control strategy which incorporates an optimized anthranilic acid feeding into a dissolved oxygen-stat (DO-stat) glucose feeding framework was proposed for the first time. Three original contributions were observed. Firstly, compared to previous DO control methods, the current strategy was able to inhibit completely the production of acetic acid, and its glucose to L-tryptophan yield reached 0.211 g/g, 62.3% higher than the previously reported. Secondly, a rigorous kinetic model was constructed to simulate the underlying biochemical process and identify the effect of anthranilic acid on both glucose conversion and L-tryptophan synthesis. Finally, a thorough investigation was conducted to testify the capability of both the kinetic model and the novel control strategy for process scale-up. It was found that the model possesses great predictive power, and the presented strategy achieved the highest glucose to L-tryptophan yield (0.224 g/g) ever reported in large scale processes, which approaches the theoretical maximum yield of 0.227 g/g. This research, therefore, paves the way to significantly enhance the profitability of the investigated bioprocess.

Biocatalytic synthesis of vitamin A palmitate using immobilized lipase produced by recombinant Pichia pastoris.

In this work, the Candida antarctica lipase B (CALB), produced by recombinant Pichia pastoris, was immobilized and used to synthesize vitamin A palmitate by transesterification of vitamin A acetate and palmitic acid in organic solvent. The reaction conditions including the type of solvent, temperature, rotation speed, particle size, and molar ratio between the two substrates were investigated. It turned out that the macroporous resin HPD826 serving as a carrier showed the highest activity (ca. 9200 U g-1) among all the screened carriers. It was found that the transesterification kinetic of the immobilized CALB followed the ping pong Bi-Bi mechanism and the reaction product acetic acid inhibited the enzymatic reaction with an inhibition factor of 2.823 mmol L-1. The conversion ability of the immobilized CALB was 54.3% after 15 cycles. In conclusion, the present work provides a green route for vitamin A palmitate production using immobilized CALB to catalyze the transesterification of vitamin A acetate and palmitic acid.

Orthogonal array deciphering MRS medium requirements for isolated Lactobacillus rhamnosus ZY with cell properties characterization.

Lactobacillus rhamnosus is a well-known lactic acid bacterium (LAB), but a new ZY strain was isolated for the first time from commercial probiotic powder recently. Although many studies have focused on developing cost-effective media for the production of LAB, the de Man, Rogosa and Sharpe (MRS) medium is still the most common medium for bioprocesses. The aim of the current study is to decipher the composition of MRS based on a statistical approach, which will allow a higher biomass of Lactobacillus to be obtained. In Taguchi's approach, an L27 orthogonal array was adopted to evaluate the significance of 10 ingredients in MRS, in which the effects of the components were ranked according to their effect on biomass at OD600 as dextrose > MnSO4·H2O > beef extract > CH3COONa > MgSO4 > yeast extract > proteose peptone > K2HPO4 > ammonium citrate > Tween 80. Although the individual trace elements of ammonium citrate, K2HPO4, CH3COONa and MgSO4 in MRS had an insignificant influence on the biomass after statistical analysis, the total elimination of trace elements would predominantly affect the cell growth of Lactobacillus. Further characterization of the cell properties through attenuated total reflectance of Fourier transform infrared (ATR-FTIR) spectroscopy and protein identification via SDS-PAGE coupled with tandem mass spectrometry implied that dextrose as major carbon source in MRS played the most crucial role for L. rhamnosus production.

Effects of lignin-derived phenolic compounds on xylitol production and key enzyme activities by a xylose utilizing yeast Candida athensensis SB18.

Candida athensensis SB18 is potential xylitol producing yeast isolated in Singapore. It has excellent xylose tolerance and is able to produce xylitol in high titer and yield. However, by-products, such as phenolic compounds, derived in lignocellulosic biomass hydrolysate might negatively influence the performance of this strain for xylitol production. In this work, four potential phenolic inhibitors, such as vanillin, syringaldehyde, 4-hydroxybenzaldehyde and phenol, were evaluated for their inhibitory effects on xylitol production by C. athensensis SB18. Phenol was shown to be the most toxic molecule on this microorganism followed by syringaldehyde. Vanillin and 4-hydroxylbenzaldehyde was less toxic than phenol and syringaldehyde, with vanillin being the least toxic. Inhibition was insignificant when the total content of inhibitors was below 1.0 g/L. The presence of phenolic compounds affected the activity of xylose reductase, however not on that of xylitol dehydrogenase. C. athensensis SB18 is therefore a potential xylitol producer from hemicellulosic hydrolysate due to its assimilation of such phenolic inhibitors.

Xylitol production from D-xylose and horticultural waste hemicellulosic hydrolysate by a new isolate of Candida athensensis SB18.

This paper describes the production of xylitol from d-xylose and horticultural waste hemicellulosic hydrolysate by a new strain of Candida athensensis SB18. Strain SB18 completely consumed 250 and 300 g L(-1) D-xylose and successful converted it to xylitol in the respective yield of 0.83 and 0.87 g g(-1), resulting in 207.8 and 256.5 g L(-1) of xylitol, respectively. The respective volumetric productivity were 1.15 and 0.97 g L(-1) h(-1). Approximately 100.1 g L(-1) of xylitol was obtained from the bioconversion of detoxified horticultural waste hemicellulosic hydrolysate using strain SB18. The yield and productivity were 0.81 g g(-1) xylose and 0.98 g L(-1) h(-1), respectively. Strain C. athensensis SB18 was able to completely utilize glucose, mannose, xylose and partially arabinose. This work demonstrates that stain C. athensensis SB18 is a promising strain for high-titer and high-yield xylitol production and it has great potential in bioconversion of hemicellulosic hydrolysate.

Enantiomer separations on a vancomycin stationary phase and retention mechanism of pressurized capillary electrochromatography.

Several chiral drugs, promethazine, carteolol, celiprolol, and albuterol, were resolved with vancomycin as the chiral stationary phase by pressurized capillary electrochromatography (pressurized CEC) and capillary HPLC. The effects of pressure and electrical field strength on efficiency, resolution, and capacity factor in pressurized CEC were investigated. A mathematical model describing the relationship of capacity factor in pressurized CEC with voltage, pressurized flow velocity, electroosmotic mobility, and electrophoretic mobility was established, which was in good agreement with the experimental data.

Separation of peptides by pressurized capillary electrochromatography.

A pressurized electrochromatography (pCEC) instrument with gradient capability was used in this work for separation of peptides. Three separation modes, namely, pCEC, high-performance liquid chromatography and capillary electrophoresiscan be carried out with the instrument. In pCEC mode, the mobile phase is driven by both electroosmotic flow and pressurized flow, facilitating fine-tuning in selectivity of neutral and charged species. A continuous gradient elution can be carried out conveniently on this instrument, which demonstrates that it is more powerful than isocratic pCEC for separation of complicated samples. The effects of applied voltage, supplementary pressure and ion-pairing agents on separation of peptides in gradient pCEC were investigated. The effects of flow-rate of the pump and the volume of the mixer on resolution were also evaluated.