High Substrate Research Articles

Structure-guided mining and engineering of nicotinamide riboside kinase for efficient synthesis of nicotinamide mononucleotide

Nicotinamide mononucleotide (NMN) is a bioactive precursor to nicotinamide adenine dinucleotide (NAD+) and has demonstrated anti-aging activity. Nicotinamide riboside kinase (NRK) can convert nicotinamide ribose (NR) into NMN in one step, but NRKs with high catalytic efficiency are rare and are difficult to engineer. In this study, two novel NRKs from Sparassis crispa and Beauveria bassiana were identified. They displayed impressive specific activities of 6.14 U/mg and 6.66 U/mg with high substrate (NR) conversion rates. Furthermore, structural modelling analysis and semi-rational design were used to develop variants of BEANRK. Variant M1 (V172K) exhibited a 1.2-fold of the wild-type BEANRK (WT) in enzyme activity and 91 % of substrate conversion rate with improved thermal stability compared to WT. Moreover, variants generated from site-specific mutagenesis were also computationally analyzed to elucidate which interactions between key amino residues and substrates facilitated an increase in catalytic activity and conversion rate. This study not only identified new NRKs but also provided insights into which amino acid residues were pivotal to the catalytic activity of NRK, paving the way for future NRK engineering to improve the industrial production of NMN.

From such great heights: The effects of substrate height and the perception of risk on lemur locomotor mechanics.

An accident during arboreal locomotion can lead to risky falls, but it remains unclear that the extent to which primates, as adept arborealists, change their locomotion in response to the perceived risk of moving on high supports in the tree canopy. By using more stable forms of locomotion on higher substrates, primates might avoid potentially fatal consequences. Using high-speed cameras, we recorded the quadrupedal locomotion of four wild lemur species-Eulemur rubriventer, Eulemur rufifrons, Hapalemur aureus, and Lemur catta (N = 113 total strides). We quantified the height, diameter, and angular orientation of locomotor supports using remote sensors and tested the influence of support parameters on gait kinematics, specifically predicting that in response to increasing substrate height, lemurs would decrease speed and stride frequency, but increase stride length and the mean number of supporting limbs. Lemurs did not adjust stride frequency on substrates of varying height. Adjustments to speed, stride length, and the mean number of supporting limbs in response to varying height often ran counter to predictions. Only E. rubriventer decreased speed and increased the mean number of supporting limbs on higher substrates. Results suggest that quadrupedal walking is a relatively safe form of locomotion for lemurs, requiring subtle changes in gait to increase stability on higher-that is, potentially riskier-substrates. Continued investigation of the impact of height on locomotion will be important to determine how animals assess risk in their environment and how they choose to use this information to move more safely.

Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%–80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.

Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine

Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S.multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.

Intensification of biocatalytic processes by using alternative reaction media

Abstract Performances of biocatalytic processes in industry are often limited by productivity, product concentration and biocatalyst stability. Reasons can be such as unfavourable reaction thermodynamics, low water solubility of the substrates or inhibition caused by high substrate or product concentrations. A way to overcome these limitations and to enhance economic competitiveness of the process can be process intensification (PI) using an alternative reaction medium. Very early in industrial biotransformation processes, it was shown that many interesting target products of organic synthesis are much more soluble and sometimes even more stable in non-conventional reaction media than in buffered aqueous solutions. Moreover, the absence of water is also generally desired to prevent side and degradation reactions as well as microbial contamination, which in turn eliminates the need to work under sterile conditions thereby reducing energy expenditure. In addition, it was also discovered early on that solvents can influence the activity and stability of enzymes quite differently depending on their water affinity and thus if they form rather monophasic or biphasic systems with the latter.

Nitrogen influence on suspended vs biofilm growth and resource recovery potential of purple non-sulfur bacteria treating fuel synthesis wastewater

Purple non-sulfur bacteria (PNSB) have a strong potential for various bioproduct avenues including single-cell protein (SCP), and production of pigments and polyhydroxybutyrate. However, high substrate and separation costs are major barriers to commercialization. Therefore, this research investigates the integration of PNSB biomass production with treatment of fuel synthesis wastewater (FSW) for a low-cost substrate and formation of biofilm for improved harvesting. An enriched PNSB culture was cultivated in two different biofilm photobioreactors (nitrogen sufficient, N+, and nitrogen-deficient, N-) with a piece of woven agricultural shade cloth as biofilm support material. Higher chemical oxygen demand (COD) removal and PNSB biofilm were obtained in the N- condition. The maximum bacteriochlorophylls occurred in the N+ suspended culture (2.65 ± 0.3 µg/g), and maximum carotenoids in the N- suspended culture (3.75 ± 1 µg/g). The cellular protein content in both conditions and biomass types was between 40 % and 44 %. The polyhydroxybutyrate content was 10–12 %, except for the N+ suspended biomass condition, which was only 3.5 %. To conclude, a nitrogen-deficient biofilm-based system showed a similar bioproduct performance to other operational modes, indicating its potential to reduce costs of PNSB-produced high value-added resources, including carotenoids, SCP, and polyhydroxybutyrate while removing organics from wastewater.

Construction of automated high-throughput screening method for finding efficient 3-ketosteroid 1,2-dehydrogenating strains.

Dehydrogenation reaction at C1(2) positions is typical and representative of industrial production of steroid drugs. Anti-inflammatory activity can be doubled when the nucleus of the anti-inflammatory steroid hormone drug introduces double bonds at the C1(2) positions. Arthrobacter simplex is currently the most widely studied and used strain for C1(2) dehydrogenation. Therefore, breeding Arthrobacter simplex with high-efficiency dehydrogenation ability is of great significance. In order to obtain high-efficiency strains, the research proposed a new screening strategy based on image process technique: firstly, a color reaction between 2,4-dinitrophenylhydrazine (DNPH) and 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) was established to characterize the dehydrogenation ability of the strain; secondly, the color data of strains mutated by atmospheric and room temperature plasma (ARTP) in the "color reaction" were automated and analyzed for dehydrogenation ability prediction using optimized support vector machine model. Result showed that the prediction accuracy reached as high as 96% in verification experiments. After a series of mutagenesis, including breaking the bottleneck of a single mutation in ARTP, the dominant strain ARLU-146 was finally obtained from 5168 strains. Its initial conversion rate was 0.8059g/L/h, with a conversion of 94.41% at 24h, compared to the original strain ASP which increased the transformation rate by more than 10%. By further process optimization, a high conversion (94.34% within 20h) with high substrate (85g/L cortisone acetate) was achieved. According to literature research, it is the highest conversion at this substrate concentration. KEY POINTS: • A high-throughput screening method was developed by using image processing and machine learning technique. • "Mutation bottleneck" of single ARTP mutagenesis was surpassed by complex mutagenesis. • A high substrate (85g/L CA) and high transformation rate craft (94.34% within 20h) were built.

Design of high SERS sensitive substrates based on branched Ti nanorods

This paper reports a rational design of branched titanium (Ti) nanorods formed by glancing angle physical vapor deposition and their applications as substrates for surface-enhanced Raman scattering (SERS). Ti nanorods with branches have larger surface areas than non-branched nanorods. However, Ti surface oxidizes easily resulting in very little SERS effect. The SERS sensitivity of the branched titanium nanorod is improved by annealing Ti nanorods in nitrogen in an effort to reduce oxidation. Additionally, the plasmonic resonance of the branched titanium nanorod is further improved by coating the top of the nanorods and branches with silver (Ag). The sensitivity of the SERS substrates is about 3700% that of as-deposited branched Ti nanorods with a native oxide layer. Our investigation provides a mechanism to fabricate sensitive SERS sensors of Ti nanorods that are known to be thermally and chemically stable and compatible with silicon-based electronics.

Effect of Clostridium butyricum Supplementation on in vitro Rumen Fermentation and Microbiota With High Grain Substrate Varying With Media pH Levels.

Clostridium butyricum (C. butyricum) can survive at low pH, and it has been widely used as an alternative to antibiotics for the improvement of feed efficiency and animal health in monogastrics. A recent study suggested that the improved ruminal fermentation with supplementing C. butyricum is may be associated with increasing the abundance of rumen microbiota in Holstein heifers, as ruminal pH plays a key role in rumen microbiota and the probiotics are often active in a dose-dependent manner. The objective of this study was to determine the effects of increasing the doses of C. butyricum on gas production (GP) kinetics, dry matter disappearance (DMD), fermentation characteristics, and rumen microbiota using a high grain substrate in batch culture varying with media pH levels. The doses of C. butyricum were supplemented at 0 (control), 0.5 × 106, 1 × 106, and 2 × 106 CFU/bottle, respectively, at either media pH 6.0 or pH 6.6. The fermentation microbiota at 0 and 1 × 106 CFU/bottle were determined using the 16S rRNA high throughput sequencing technology. Overall, the GP, DMD, total volatile fatty acid (VFA) concentration, and the ratio of acetate:propionate were higher (P <0.01) at media pH 6.6 than at pH 6.0. However, there was interaction between pH × dose of C. butyricum for rate constant of GP (P = 0.01), average GP rate (P = 0.07), and volume of GP (P = 0.06); with the increase in C. butyricum supplementation, the GP kinetics were not changed at media pH 6.0, but the volume (P = 0.02), rate of GP (P = 0.01), and average GP rate (P = 0.01) were quadratically changed at media pH 6.6. The DMD was not affected by increasing the supplementation of C. butyricum. The molar proportions of propionate (P <0.09), butyrate (P <0.06), and NH3-N concentration (P = 0.02) were quadratically changed with increasing supplementation of C. butyricum regardless of media pH levels. The interactions between media pH level and dose of C. butyricum supplementation were noticed for alpha diversity indexes of Shannon (P = 0.02) and Evenness (P = 0.04). The alpha diversity indexes increased (P <0.05) except for Chao1 with supplementation of C. butyricum. The unweighted uniFrac analysis showed that the group of control at media pH 6.0 and control at media pH 6.6, and supplementation of C. butyricum and control at media pH 6.0 clustered separately from each other. At the phylum level, relative abundance (RA) of Bacteroidota was lower (P <0.01) and Firmicutes was higher (P <0.01) at media pH 6.6 than pH 6.0. Moreover, RA of Proteobacteria decreased (P <0.05) with supplemented C. butyricum at either media pH 6.6 or pH 6.0. At media pH 6.6, RA of Rikenellaceae_RC9_gut_group and Prevotella were decreased, and CAG-352 was increased (at genus level) compared to pH 6.0. Supplementation of C. butyricum decreased RA of Rikenellaceae_RC9_gut_group and increased CAG-352 at media pH 6.0. It could hence be concluded that manipulating media pH level and supplementation of C. butyricum effectively modulated in vitro rumen fermentation characteristics and microbiota but in a dose depending manner of C. butyricum addition.

Computer-Aided Design of α-L-Rhamnosidase to Increase the Synthesis Efficiency of Icariside I.

Icariside I, the glycosylation product of icaritin, is a novel effective anti-cancer agent with immunological anti-tumor activity. However, very limited natural icariside I content hinders its direct extraction from plants. Therefore, we employed a computer-aided protein design strategy to improve the catalytic efficiency and substrate specificity of the α-L-rhamnosidase from Thermotoga petrophila DSM 13995, to provide a highly-efficient preparation method. Several beneficial mutants were obtained by expanding the active cavity. The catalytic efficiencies of all mutants were improved 16–200-fold compared with the wild-type TpeRha. The double-point mutant DH was the best mutant and showed the highest catalytic efficiency (k cat /K M : 193.52 s−1 M−1) against icariin, which was a 209.76-fold increase compared with the wild-type TpeRha. Besides, the single-point mutant H570A showed higher substrate specificity than that of the wild-type TpeRha in hydrolysis of different substrates. This study provides enzyme design strategies and principles for the hydrolysis of rhamnosyl natural products.

Engineering the Active Site Pocket to Enhance the Catalytic Efficiency of a Novel Feruloyl Esterase Derived From Human Intestinal Bacteria Dorea formicigenerans

The human gut microbiota play essential roles in metabolism and human health, especially by enzymatically utilizing dietary fiber that the host cannot directly digest and releasing functional components including short-chain fatty acids (SCFAs) and hydroxycinnamic acids (e.g., ferulic acid). In our previous study, seven potential feruloyl esterase (FAE) genes were identified from the gut microbiota. In the current work, one of the genes encoding a novel FAE (DfFAE) from Dorea formicigenerans of Firmicutes was bacterially expressed, purified and characterized. The 30.5 kDa type-A DfFAE has an optimum pH and temperature of 8.4 and 40 °C, respectively, exhibiting a higher substrate specificity toward short-chain acyl-ester substrate (pNPA). The AlphaFold2 based ab initio structural modeling revealed a five α-helices cap domain that shaped an unusually narrow and deep active site pocket containing a specific substrate access tunnel in DfFAE. Furthermore, rational design strategy was subjected to the active site pocket in an aim of improving its enzymatic activities. The mutants V252A, N156A, W255A, P149A, and P186A showed 1.8 to 5.7-fold increase in catalytic efficiency toward pNPA, while W255A also exhibited altered substrate preference toward long-chain substrate pNPO (45.5-fold). This study highlighted an unusual active site architecture in DfFAE that influenced its substrate selectivity and illustrated the applicability of rational design for enhanced enzymatic properties.

Laboratory research of solvent-assisted menthol sols as temporary consolidants in archaeological excavation applications

Temporary consolidation is a quite common practice during archeological excavations nowadays. Melts of cyclododecane or menthol are among the most used consolidants. When using melting protocol to consolidate fragile heritages in archaeological excavation sites, one of the most frequently encountered challenges is the poor penetration of the melt into the heritages, especially in cases of low environmental temperatures, high water content or compact substrates. In this work, we explore the possibility of using solvent-assisted menthol sols as temporary consolidant. Six common organic liquids are individually introduced into menthol at a concentration of 9.1 wt% to formulate room temperature menthol sols. Their potentials as temporary consolidants are systematically investigated. Experimental data indicate that solvent polarity is the most important feature for temporary consolidation purpose and ethanol with medium polarity is among the most appropriate solvents. Laboratory research results show that much better penetration behavior and good consolidation performances can be achieved in menthol-ethanol sol. The as-prepared menthol-ethanol sol is applied in Liangzhu archeological excavation site with satisfactory outcomes. This work shows that menthol-ethanol sol is an excellent temporary consolidation material for archaeological excavation purpose especially in extremely wet condition.

Multiple Tanks Technique: Application to silicon-based radio-frequency and millimeter-wave integrated circuits

Radio-frequency (RF) integrated circuits (ICs) and millimeter-wave (mm-wave) ICs play crucial roles in modern wireless communication systems in laptops, smart phones, tablets, and so on. Silicon-based processes that have low cost and high integrity prompt the wide adoption of RF/mm-wave ICs in consumer electronics. However, the high substrate and metal losses of commercial silicon are considered inherent drawbacks, especially for RFIC designs with low-resistivity silicon of ∼10 Ω/square. We propose a multiple tanks topology to overcome this issue. The topology utilizes coupled multiple coils as a transformer to improve the equivalent quality factor (Q factor) of the tanks. Different applications if ICs, such as voltage-controlled oscillators (VCOs), dividers, power amplifiers (PAs), switches, and other circuits with frequencies up to 300 GHz, verify that by using this technique, dedicated RF/mm-wave IC performance can be significantly improved. Moreover, a 60-GHz transceiver system-on-chip (SOC) based on this technique is verified. This article presents the proposed method and implementation, from the fundamental concept and analysis to IC and system verification.

High Strength Ceramic Substrates Based on Perlite and Foam Silicates for Filtration Membranes

High Strength Ceramic Substrates Based on Perlite and Foam Silicates for Filtration Membranes

AlGaN/GaN High Electron Mobility Transistors Grown by MOVPE on 3C-SiC/Si(111) for RF Applications

In this work, an AlGaN/GaN HEMT structure is grown on a 0.8 μm thick 3C-SiC layer on high resistivity Silicon substrate. The RF propagation losses are investigated and compared with the ones of epi-layers grown directly on Silicon and on 6H-SiC substrates. Short gate length transistors are fabricated using e-beam lithography. In spite of ohmic contact resistance of 0.6 Ω.mm, a saturated current density of 0.7 A/mm at a gate bias of +1V and a transconductance peak higher to 250 mS/mm for 75 nm T-shaped gate transistors are reached on structure with thick 3C-SiC template. Moreover, for the first time, transition frequencies fT/fmax of 60/98 GHz are reported on such 3C-SiC template.

Modulation of the 20S Proteasome Activity by Porphyrin Derivatives Is Steered through Their Charge Distribution.

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a “new” 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure–activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg–Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a “contracted” version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis.

SulfoxFluor-enabled deoxyazidation of alcohols with NaN3

Direct deoxyazidation of alcohols with NaN3 is a straightforward method for the synthesis of widely used alkyl azides in organic chemistry. However, known methods have some limitations such as high reaction temperatures and narrow substrate scope. Herein, a general and practical method for the preparation of alkyl azides from alcohols using NaN3 has been developed. N-tosyl-4-chlorobenzenesulfonimidoyl fluoride (SulfoxFluor) plays an important role in this deoxyazidation process, which converts a broad range of alcohols into alkyl azides at room temperature. The power of this deoxyazidation protocol has been demonstrated by successful late-stage deoxyazidation of natural products and pharmaceutically relevant molecules.

HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3’UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.

Transport efficiency of AtGTR1 dependents on the hydrophobicity of transported glucosinolates

Glucosinolates (GLSs) are a group of secondary metabolites that are involved in the defense of herbivores. In Arabidopsis thaliana, Glucosinolate Transporter 1 (AtGTR1) transports GLSs with high affinity via a proton gradient-driven process. In addition to transporting GLSs, AtGTR1 also transports phytohormones, jasmonic acid-isoleucine (JA-Ile), and gibberellin (GA). However, little is known about the mechanisms underlying the broad substrate specificity of AtGTR1. Here, we characterized the substrate preference of AtGTR1 by using a yeast uptake assay, and the results revealed that GLS transport rates are negatively correlated with the hydrophobicity of substrates. Interestingly, the AtGTR1 showed a higher substrate affinity for GLSs with higher hydrophobicity, suggesting a hydrophobic substrate binding pocket. In addition, competition assays revealed that JA, salicylic acid (SA), and indole-3-acetic acid (IAA) competed with GLS for transport in yeast, suggesting a potential interaction of AtGTR1 with these phytohormones. To further characterize the functional properties of AtGTR1, mutagenesis experiments confirmed that the conserved EXXEK motif and Arg166 are essential for the GLS transport function. In addition, the purified AtGTR1 adopts a homodimeric conformation, which is possibly regulated by phosphorylation on Thr105. The phosphomimetic mutation, T105D, reduced its protein expression and completely abrogated its GLS transport function, indicating the essential role of phosphorylation on AtGTR1. In summary, this study investigated various factors associated with the GLS transport and increased our knowledge on the substrate preferences of AtGTR1. These findings contribute to understanding how the distribution of defense GLSs is regulated in plants and could be used to improve crop quality in agriculture.

Declining carbohydrate solubilization with increasing solids loading during fermentation of cellulosic feedstocks by Clostridium thermocellum: documentation\xa0and diagnostic tests

BackgroundFor economically viable 2nd generation biofuels, processing of high solid lignocellulosic substrate concentrations is a necessity. The cellulolytic thermophilic anaerobe Clostridium thermocellum is one of the most effective biocatalysts for solubilization of carbohydrate harbored in lignocellulose. This study aims to document the solubilization performance of Clostridium thermocellum at increasing solids concentrations for two lignocellulosic feedstocks, corn stover and switchgrass, and explore potential effectors of solubilization performance.ResultsMonocultures of Clostridium thermocellum demonstrated high levels of carbohydrate solubilization for both unpretreated corn stover and switchgrass. However, fractional carbohydrate solubilization decreases with increasing solid loadings. Fermentation of model insoluble substrate (cellulose) in the presence of high solids lignocellulosic spent broth is temporarily affected but not model soluble substrate (cellobiose) fermentations. Mid-fermentation addition of cells (C. thermocellum) or model substrates did not significantly enhance overall corn stover solubilization loaded at 80 g/L, however cultures utilized the model substrates in the presence of high concentrations of corn stover. An increase in corn stover solubilization was observed when water was added, effectively diluting the solids concentration mid-fermentation. Introduction of a hemicellulose-utilizing coculture partner, Thermoanaerobacterium thermosaccharolyticum, increased the fractional carbohydrate solubilization at both high and low solid loadings. Residual solubilized carbohydrates diminished significantly in the presence of T. thermosaccharolyticum compared to monocultures of C. thermocellum, yet a small fraction of solubilized oligosaccharides of both C5 and C6 sugars remained unutilized.ConclusionDiminishing fractional carbohydrate solubilization with increasing substrate loading was observed for C. thermocellum-mediated solubilization and fermentation of unpretreated lignocellulose feedstocks. Results of experiments involving spent broth addition do not support a major role for inhibitors present in the liquid phase. Mid-fermentation addition experiments confirm that C. thermocellum and its enzymes remain capable of converting model substrates during the middle of high solids lignocellulose fermentation. An increase in fractional carbohydrate solubilization was made possible by (1) mid-fermentation solid loading dilutions and (2) coculturing C. thermocellum with T. thermosaccharolyticum, which ferments solubilized hemicellulose. Incomplete utilization of solubilized carbohydrates suggests that a small fraction of the carbohydrates is unaffected by the extracellular carbohydrate-active enzymes present in the culture.