Tuning Structural Characteristics of Corn Stover Through Ammonium and Sodium Sulfite (ASS) Pretreatment for Enhanced Enzymatic Hydrolysis.

The ammonia fiber expansion (AFEX) pretreatment of lignocellulosic biomass offers a significant advantage in terms of obtaining high glucan conversion, with the added benefit of ammonia being fully recyclable. However, despite the high efficiency of AFEX in pretreating lignocellulose, relatively high enzyme loading is still required for effective cellulose conversions. In this study, we have updated the AFEX pretreatment method; ammonia and sodium sulfite (ASS) can be used to produce a more digestible substrate. The results demonstrate that ASS-pretreated corn stover (CS) yields a higher fermentable sugar yield compared with AFEX pretreatment, even at lower enzyme loadings. Specifically, at an enzyme loading of 12 mg protein/g glucan, ASS-CS achieved 88.8% glucose and 80.6% xylose yield. Characterization analysis reveals that lignin underwent sulfonation during ASS pretreatment. This modification results in a more negative zeta potential for ASS-CS, indicating a reduction in nonproductive adsorption between lignin and cellulase through increased electrostatic repulsion.

Isomerase and epimerase: overview and practical application in production of functional sugars.

The biosynthesis of functional sugars has gained significant attention due to their potential health benefits and increasing demand in the food industry. Enzymatic synthesis has emerged as a promising approach, offering high catalytic efficiency, chemoselectivity, and stereoselectivity. However, challenges such as poor thermostability, low catalytic efficiency, and food safety concerns have limited the commercial production of functional sugars. Protein engineering, including directed evolution and rational design, has shown promise in overcoming these barriers and improving biocatalysts for large-scale production. Furthermore, enzyme immobilization has proven effective in reducing costs and facilitating the production of functional sugars. To ensure food safety, the use of food-grade expression systems has been explored. However, downstream technologies, including separation, purification, and crystallization, still pose challenges in terms of efficiency and cost-effectiveness. Addressing these challenges is crucial to optimize the overall production process. Despite the obstacles, the future outlook for functional sugars is promising, driven by increasing awareness of their health benefits and continuous technological advancements. With further research and technological breakthroughs, industrial-scale production of functional sugars through biosynthesis will become a reality, leading to their widespread incorporation in various industries and products.

A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination.

BACKGROUND: Budding yeast, Saccharomyces cerevisiae, has been extensively favored as a model organism in aging and age-related studies, thanks to versatile microfluidic chips for cell dynamics assay and replicative lifespan (RLS) determination at single-cell resolution. However, previous microfluidic structures aiming to immobilize haploid yeast may impose excessive spatial constraint and mechanical stress on cells, especially for larger diploid cells that sprout in a bipolar pattern. RESULTS: We developed a high-throughput microfluidic chip for diploid yeast long-term culturing (DYLC), optical inspection and cell-aging analysis. The DYLC chip features 1100 "leaky bowl"-shaped traps formatted in an array to dock single cells under laminar-perfused medium and effectively remove daughter cells by hydraulic shear forces. The delicate microstructures of cell traps enable hydrodynamic rotation of newborn buds, so as to ensure bud reorientation towards downstream and concerted daughter dissection thereafter. The traps provide sufficient space for cell-volume enlargement during aging, and thus properly alleviate structural compression and external stress on budding yeast. Trapping efficiency and long-term maintenance of single cells were optimized according to computational fluid dynamics simulations and experimental characterization in terms of critical parameters of the trap and array geometries. Owing to the self-filling of daughter cells dissected from traps upstream, an initial trapping efficiency of about 70% can rapidly reach a high value of over 92% after 4-hour cell culturing. During yeast proliferation and aging, cellular processes of growth, budding and daughter dissection were continuously tracked for over 60 h by time-lapse imaging. Yeast RLS and budding time interval (BTI) were directly calculated by the sequential two-digit codes indicating the budding status in images. With the employed diploid yeast strain, we obtained an RLS of 24.29 ± 3.65 generations, and verified the extension of BTI in the first couple of generations after birth and the last several generations approaching death, as well as cell de-synchronization along diploid yeast aging. CONCLUSIONS: The DYLC chip offers a promising platform for reliable capture and culturing of diploid yeast cells and for life-long tracking of cell dynamics and replicative aging processes so that grasping comprehensive insights of aging mechanism in complex eukaryotic cells.

Real-Time Monitoring of Dissection Events of Single Budding Yeast in a Microfluidic Cell-Culturing Device Integrated With Electrical Impedance Biosensor.

Microfluidic devices in combination with fluorescent microscopy offer high-resolution and high-content platforms to study single-cell morphology, behavior and dynamic process in replicative aging of budding yeast, Saccharomyces cerevisiae. However, a huge mass of recorded images makes the data processing labor-intensive and time-consuming to determine yeast replicative lifespan (RLS), a primary criterion in yeast aging. To address this limitation and pursue label-free RLS assays, electrical impedance spectroscopy (EIS) that can be easily functionalized through microelectrodes in microfluidic devices, was introduced to monitor cell growth and division of budding yeast. Herein, a microfluidic device integrated with EIS biosensor was proposed to perform in-situ impedance measurement of yeast proliferation in single-cell resolution so as to identify the momentary events of daughter dissection from its mother. Single yeast cells were reliably immobilized at the bottleneck-like traps for continuous culturing, during which daughter cells were effectively detached from their mother cells by hydraulic shear forces. Time-lapse impedance measurement was performed every 2 min to monitor the cellular process including budding, division and dissection. By using the K-means clustering algorithm to analyze a self-defined parameter "Dissection Indicator," to our knowledge for the first time, the momentary event of a daughter removing from its mother cell was accurately extracted from EIS signals. Thus, the identification of daughter dissection events based on impedance sensing technology has been validated. With further development, this microfluidic device integrated with electrical impedance biosensor holds promising applications in high-throughput, real-time and label-free analysis of budding yeast aging and RLS.

Efficient lactic acid production from dilute acid-pretreated lignocellulosic biomass by a synthetic consortium of engineered Pseudomonas putida and Bacillus coagulans.

BACKGROUND: Lignocellulosic biomass is an attractive and sustainable alternative to petroleum-based feedstock for the production of a range of biochemicals, and pretreatment is generally regarded as indispensable for its biorefinery. However, various inhibitors that severely hinder the growth and fermentation of microorganisms are inevitably produced during the pretreatment of lignocellulose. Presently, there are few reports on a single microorganism that can detoxify or tolerate toxic mixtures of pretreated lignocellulose hydrolysate while effectively transforming sugar components into valuable compounds. Alternatively, microbial coculture provides a simpler and more efficacious way to realize this goal by distributing metabolic functions among different specialized strains. RESULTS: In this study, a novel synthetic microbial consortium, which is composed of a responsible for detoxification bacterium engineered Pseudomonas putida KT2440 and a lactic acid production specialist Bacillus coagulans NL01, was developed to directly produce lactic acid from highly toxic lignocellulosic hydrolysate. The engineered P. putida with deletion of the sugar metabolism pathway was unable to consume the major fermentable sugars of lignocellulosic hydrolysate but exhibited great tolerance to 10 g/L sodium acetate, 5 g/L levulinic acid, 10 mM furfural and HMF as well as 2 g/L monophenol compound. In addition, the engineered strain rapidly removed diverse inhibitors of real hydrolysate. The degradation rate of organic acids (acetate, levulinic acid) and the conversion rate of furan aldehyde were both 100%, and the removal rate of most monoaromatic compounds remained at approximately 90%. With detoxification using engineered P. putida for 24 h, the 30% (v/v) hydrolysate was fermented to 35.8 g/L lactic acid by B. coagulans with a lactic acid yield of 0.8 g/g total sugars. Compared with that of the single culture of B. coagulans without lactic acid production, the fermentation performance of microbial coculture was significantly improved. CONCLUSIONS: The microbial coculture system constructed in this study demonstrated the strong potential of the process for the biosynthesis of valuable products from lignocellulosic hydrolysates containing high concentrations of complex inhibitors by specifically recruiting consortia of robust microorganisms with desirable characteristics and also provided a feasible and attractive method for the bioconversion of lignocellulosic biomass to other value-added biochemicals.

A microfluidic single-cell array for in situ laminar-flow-based comparative culturing of budding yeast cells.

To facilitate in situ comparative culturing of budding yeast cells in a precisely controlled microenvironment, we developed a microfluidic single-cell array (MiSCA) with 96 traps (16 rows × 6 columns) for single-cell immobilization. Through optimization of the distances between neighboring traps and the applied flow rates by using a hydraulic equivalent circuit of the fluidic network, yeast cells were delivered to each column of the array by laminar focused flows and reliably captured at the traps by hydrodynamic forces with about 90% efficiency of cell immobilization. Immobilized cells in different columns within the same device can then be cultured in parallel while being exposed to different media and compounds delivered by laminar flows. For biological validation of the comparative cell-culturing device, we used budding yeast that can express yellow fluorescent protein upon the addition of ß-estradiol in cell-culturing medium. Experimental results show successful induction of fluorescence in cells immobilized in desired columns that have been dosed with ß-estradiol. The MiSCA system allows for performing sets of experiments and control experiments in parallel in the same device, or for executing comparative experiments under well-defined laminar-perfusion conditions with different media, as well as in situ monitoring of dynamic cellular responses upon different analytical compounds or reagents for single-cell analysis.

Comprehensive utilization of corncob for furfuryl alcohol production by chemo-enzymatic sequential catalysis in a biphasic system.

A new process for the production of furfuryl alcohol from corncob was constructed by using deep eutectic solvents and whole cell catalysis in this paper. Firstly, the corncob was treated with deep eutectic solvents to convert the xylan into furfural, and then the pretreated corncob residue was enzymatically hydrolyzed to obtain a glucose-rich enzymatic hydrolysate, which was used to provide NADH for Bacillus coagulans NL01 during the process of furfural reduction. The furfural yield could reach 46% using the selected choline chloride-oxalic acid as catalysts and corncob as substrate under the optimized catalytic condition at 120 °C for 30 min. The final furfuryl alcohol yield of 20.7% was achieved with corncob as substrate. Moreover, this catalytic system realized the recycling of deep eutectic solvents three times, the high-value production of furfuryl alcohol, and the comprehensive utilization of corncob.

The key role of delignification in overcoming the inherent recalcitrance of Chinese fir for biorefining.

The enzymatic digestibility of softwood is hindered for its highly recalcitrant nature to enzymatic attack. In this study, the effects of dilute sulfuric acid pretreatment (DSAP), acidic sodium chlorite pretreatment (SCP), and their combined pretreatments (DSA-SCP and SC-DSAP) on Chinese fir sawdust were investigated, respectively. Results demonstrated that lignin was the most important obstacle, and digestibility increased linearly with lignin removal yield. Furthermore, the results revealed that the order of sequential pretreatment significantly affected the delignification, and hemicellulose should be removed first. Compared to SC-DSAP, DSA-SCP involving the hemicellulose-removal-first strategy exhibited higher delignification efficiency. DSA-SCP caused lignin removal of 92.3% and the enzymatic hydrolysis was high of 97.9%. Finally, a regression model with high reliability was established to quickly evaluate pretreatment process. In summary, this study highlighted the importance of delignification for saccharification of softwood and unveiled the effect of hemicellulose on delignification.

One-pot process for lactic acid production from wheat straw by an adapted Bacillus coagulans and identification of genes related to hydrolysate-tolerance.

In this study, Bacillus coagulans CC17A with highly tolerant to hydrolysate was obtained through adaptive evolution. After 63 generations, the strain CC17A was stably in 45% (v/v) hydrolysate media and could digest multiple inhibitors in the hydrolysate. Based on its promising features, a one-pot process was developed to produce lactic acid (LA) from wheat straw. After dilute acid pretreatment of wheat straw, simultaneous saccharification and co-fermentation was conducted using CC17A without any solid-liquid separation and pre-detoxification. Total 35.50 g LA was produced from 80 g raw substrate and the production yield was as high as 70.9% of theoretical. To elucidate the tolerance mechanism, transcriptomic profiling of CC17A was studied. The highly up-regulated oxidoreductases and phenolic acid decarboxylase are considered to be involved with the inhibitors-tolerance of B. coagulans CC17A.

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device.

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single-cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 µm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 µm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP-release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single-cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP-release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single-cell analysis.

Simultaneous consumption of cellobiose and xylose by Bacillus coagulans to circumvent glucose repression and identification of its cellobiose-assimilating operons.

BACKGROUND: The use of inedible lignocellulosic biomasses for biomanufacturing provides important environmental and economic benefits for society. Efficient co-utilization of lignocellulosic biomass-derived sugars, primarily glucose and xylose, is critical for the viability of lignocellulosic biorefineries. However, the phenomenon of glucose repression prevents co-utilization of both glucose and xylose in cellulosic hydrolysates. RESULTS: To circumvent glucose repression, co-utilization of cellobiose and xylose by Bacillus coagulans NL01 was investigated. During co-fermentation of cellobiose and xylose, B. coagulans NL01 simultaneously consumed the sugar mixtures and exhibited an improved lactic acid yield compared with co-fermentation of glucose and xylose. Moreover, the cellobiose metabolism of B. coagulans NL01 was investigated for the first time. Based on comparative genomic analysis, two gene clusters that encode two different operons of the cellobiose-specific phosphoenolpyruvate-dependent phosphotransferase system (assigned as CELO1 and CELO2) were identified. For CELO1, five genes were arranged as celA (encoding EIIAcel), celB (encoding EIIBcel), celC (encoding EIICcel), pbgl (encoding 6-phospho-ß-glucosidase), and celR (encoding a transcriptional regulator), and these genes were found to be ubiquitous in different B. coagulans strains. Based on gene knockout results, CELO1 was confirmed to be responsible for the transport and assimilation of cellobiose. For CELO2, the five genes were arranged as celR, celB, celA, celX (encoding DUF871 domain-containing protein), and celC, and these genes were only found in some B. coagulans strains. However, through a comparison of cellobiose fermentation by NL01 and DSM1 that only possess CELO1, it was observed that CELO2 might also play an important role in the utilization of cellobiose in vivo despite the fact that no pbgl gene was found. When CELO1 or CELO2 was expressed in Escherichia coli, the recombinant strain exhibited distinct cellobiose uptake and consumption. CONCLUSIONS: This study demonstrated the cellobiose-assimilating pathway of B. coagulans and provided a new co-utilization strategy of cellobiose and xylose to overcome the obstacles that result from glucose repression in a biorefinery system.

Fumaric Acid Production from Alkali-Pretreated Corncob by Fed-Batch Simultaneous Saccharification and Fermentation Combined with Separated Hydrolysis and Fermentation at High Solids Loading.

Production of fumaric acid from alkali-pretreated corncob (APC) at high solids loading was investigated using a combination of separated hydrolysis and fermentation (SHF) and fed-batch simultaneous saccharification and fermentation (SSF) by Rhizopus oryzae. Four different fermentation modes were tested to maximize fumaric acid concentration at high solids loading. The highest concentration of 41.32 g/L fumaric acid was obtained from 20 % (w/v) APC at 38 °C in the combined SHF and fed-batch SSF process, compared with 19.13 g/L fumaric acid in batch SSF alone. The results indicated that a combination of SHF and fed-batch SSF significantly improved production of fumaric acid from lignocellulose by R. oryzae than that achieved with batch SSF at high solids loading.