High Substrate Loading Research Articles

High-temperature tolerant enzyme based extraction of lignocellulose derived C5 and C6 sugars for the integrated production of a promising supplement for a diesel/jet-engine fuel

Development of a novel, integrated production of C8 ester butyl butyrate (BuB), a promising supplement for diesel/jet-engine fuels, from lignocellulose derived C5 and C6 sugars is attempted by means of smart and innovative process design. In the pathway, enzymatic hydrolysis of biomass with high solid loading is an important step which requires reduced amount of water supply and energy input. However, increased substrate loading lead to diffusion limitations of enzymes, on top of nonproductive absorption of enzymes. Addition of ß-glucosidase over cellulase is another reason to increase the cost of enzymes. Viscozyme can be considered as a troubleshooter in these cases where it can offset high substrate loading and increased monomeric sugar generation. In the present study, 17.61 g/L of glucose, along with 7.43 g/L of xylose, have been recovered with a viscozyme concentration of 54.4 g/L. Additionally, thermal deactivation of viscozyme has been investigated. The deactivation constant is found to be gradually increasing with escalated enzyme loading from 24.2 g/L to 54.4 g/L. In this study, it has been found that hydrolysis of biomass follows a first order pseudo-kinetics and the same is eventually used to determine the fractal kinetics of cellulose hydrolysis. Fractal kinetics showed that increment of enzyme concentration is associated with lower fractal dimensions and increased binding capacity of substrate and enzyme. Diffusion limitation of the present enzyme-substrate system has also been investigated. It is observed that faster diffusion, along with increased resistances, can be achieved with increased enzyme concentration.

Efficient biosynthesis of (R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol using a mutant short-chain dehydrogenase from Novosphingobium aromaticivorans

(R)-2-chloro-1-(2, 4-dichlorophenyl) ethanol ((R)-CPEO) is an important chiral intermediate for antifungal drug synthesis. (R)-CPEO can be produced from 2-chloro-1-(2, 4-dichlorophenyl) ethanone (CPE) via a mutant short-chain dehydrogenase/reductase from Novosphingobium aromaticivorans (NaSDR). The Vmax of a mutant NaSDR-G145A/I199L toward CPE (6.32 U mg−1) was greater than that of wild-type NaSDR (2.58 U mg−). The Km of mutant NaSDR-G145A/I199L toward CPE (0.23 mM) was less than that of wild-type NaSDR (0.38 mM), indicating that the substrate affinity of mutant NaSDR-G145A/I199L was the greater of the two. Docking simulations were used to illustrate the mechanisms of the increased enzyme activity of NaSDR-G145A/I199L; these showed that NaSDR-G145A/I199L presented a more effective docking posture than that of the wild-type enzyme. Further, NaSDR-G145A/I199L and glucose dehydrogenase (GDH) were used to transform 120 g/L CPE into (R)-CPEO. After 6 h, the conversion rate and enantiomeric excess values were 99% and 99.95%, respectively. The present study provides a practical method for high substrate loading of (R)-CPEO for industrial-scale applications.

Design and evaluation of novel bio‐based deep eutectic solvents for highly efficient bioproduction of chiral aryl alcohol

AbstractBACKGROUNDDeep eutectic solvents (DESs) derived from bio‐available materials are promising eco‐friendly solvents in the field of biocatalysis. However, the type of bio‐based DES is still limited and its application in bioprocess has not been well‐explored. We adopted a design strategy via the combination of choline and amino acids to synthesize novel bio‐based DESs, aimed to enhance biocatalytic efficacy by exploiting the developed DESs as co‐solvents.RESULTSVarious bio‐based DESs were synthesized, where the two components are both bio‐available (choline and amino acids). Their performances in the bioreduction of 2‐chloro‐1‐(3,4‐difluorophenyl)ethanone (CFPO) were subsequently evaluated with recombinant Escherichia coli cells. The efficient asymmetric production of (S)‐2‐chloro‐1‐(3,4‐difluorophenyl)ethanol ((S)‐CFPL) was successfully achieved in a developed choline acetate/lysine (ChAc/Lys)‐containing medium. ChAc/Lys can strengthen coenzyme regeneration and properly improve cell membrane permeability during bioreduction, which account for high conversion. After optimized reaction parameters were conducted in the ChAc/Lys‐buffer system, 87.0% (S)‐CFPL yield was observed with > 99.9% enantiomeric excess (ee) value at 1 mol L−1 CFPO content. The substrate loading in the established ChAc/Lys‐buffer system was increased by 3.3‐fold compared to aqueous medium. This bioprocess was also proved to be feasible at 500 mL preparative‐scale with a satisfactory conversion.CONCLUSIONChAc/Lys bio‐DES is a promising and biocompatible co‐solvent. Introducing ChAc/Lys to the reaction system is an effective strategy for challenging high substrate loading for bioreduction. Our results give a valuable insight for developing green and benign sustainable bio‐DESs for biotransformation. © 2020 Society of Chemical Industry

Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective "Michaelase" for Catalysis in High Concentrations of Ethanol.

Enzymes have evolved to function under aqueous conditions and may not exhibit features essential for biocatalytic application, such as the ability to function in high concentrations of an organic solvent. Consequently, protein engineering is often required to tune an enzyme for catalysis in non‐aqueous solvents. In this study, we have used a collection of nearly all single mutants of 4‐oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful Michael‐type additions of acetaldehyde to various nitroolefins, to investigate the effect of each mutation on the ability of this enzyme to retain its “Michaelase” activity in elevated concentrations of ethanol. Examination of this mutability landscape allowed the identification of two hotspot positions, Ser30 and Ala33, at which mutations are beneficial for catalysis in high ethanol concentrations. The “hotspot” position Ala33 was then randomized in a highly enantioselective, but ethanol‐sensitive 4‐OT variant (L8F/M45Y/F50A) to generate an improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40 % ethanol (permitting high substrate loading) to give the desired γ‐nitroaldehyde product in excellent isolated yield (89 %) and enantiopurity (ee=98 %). The presented work demonstrates the power of mutability‐landscape‐guided enzyme engineering for efficient biocatalysis in non‐aqueous solvents.

Efficient Formation of 2,5-Diformylfuran in Ionic Liquids at High Substrate Loadings and Low Oxygen Pressure with Separation through Sublimation

The oxidation of 5-hydroxymethylfurfural (HMF) to 2,5-diformylfuran (DFF) using oxygen (1 atm) with a TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl) and CuCl catalyst system is investigated using ...

Efficient asymmetric synthesis of chiral alcohols using high 2-propanol tolerance alcohol dehydrogenase SmADH2 via an environmentally friendly TBCR system

Based on substrate-coupled cofactor regeneration system, a high 2-propanol tolerance SmADH2 together with TBCR system can synthesise structurally diverse chiral alcohols at a high substrate loading with only 1.25 equivalents of 2-propanol.

Genetics of Unstudied Thermophiles for Industry.

Thermophilic organisms hold great potential for industry due to their numerous advantages in biotechnological applications such as higher reaction rate, higher substrate loading, decreased susceptibility to reaction contamination, energy savings in industrial fermentations, and ability to express thermostable proteins that can be utilized in many important industrial processes. Bioprospecting for thermophiles will continue to reveal new enzymatic and metabolic paradigms with industrial applicability. In order to translate these paradigms to production scale, routine methods for microbial genetic engineering are needed, yet remain to be developed in many newly isolated thermophiles. Major challenges and recent developments in the establishment of reliable genetic systems in thermophiles are discussed. Here, we use a hyperthermophilic, cellulolytic bacterium, Caldicellulosiruptor bescii, as a case study to demonstrate the development of a genetic system for an industrially useful thermophile, describing in detail methods for transformation, genetic tool utilization, and chromosomal modification using targeted gene deletion and insertion techniques.

Factors influencing volatile fatty acids production from food wastes via anaerobic digestion

ABSTRACT Volatile fatty acids (VFAs) are intermediate products in anaerobic digestion. The effect of substrate loading or inoculum to substrate ratio (ISR), the addition of methanogen inhibitor, O2 presence, control the reactor’s pH, and inoculum adaptation on the VFAs production from food waste through acidogenesis process was investigated in this study. Addition of 2-bromoethane sulfonic (BES) as methanogen inhibitor suppressed VFA consumption by methanogens at ISR 1:1. At higher substrate loading (ISR 1:3), methane production can be suppressed even without the addition of BES. However, at high substrate loading, controlling the pH during acidogenesis is important to achieve high VFAs yield. Acclimatization of inoculum is also one of the strategies to achieve high VFA yield. The highest VFAs yield obtained in this work was 0.8 g VFA/g VS added at ISR 1:3, controlled pH at 6, with the presence of initial O2 (headspace unflushed).

Ethanol production from banana peels at high pretreated substrate loading: comparison of two operational strategies

The potential of banana peels (BPs) as a source of fermentable sugars for ethanol production was evaluated using two yeasts: Saccharomyces cerevisiae commercial strain and Kluyveromyces marxianus thermotolerant strain isolated from Agave honey. Different process configurations: simultaneous saccharification and fermentation (SSF), as well as presaccharification and simultaneous saccharification and fermentation (PSSF) at high solid loading (up to 25% w/w), were studied. Similar ethanol production 13 g/L and 11 g/L was achieved at 10–12 h on SSF for S. cerevisiae and K. marxianus at 35 and 41 °C, respectively, with 10% (w/w) BP. The highest ethanol concentration (32.6 g/L) was reached on PSSF process (at 64 h) using commercial S. cerevisiae strain with 25% (w/v) BP, while ethanol production was inhibited in the case of K. marxianus under same assay conditions. BPs are a suitable source for second-generation ethanol production. The advantage of S. cerevisiae in the PSSF in comparison with K. marxianus was demonstrated. The present study demonstrated that BPs are the promising feedstock for ethanol production by PSSF.

Bio-hydrogen production from tequila vinasses: Effect of detoxification with activated charcoal on dark fermentation performance

Hydrogen production from dark fermentation is a potential source of sustainable fuel when it is generated from waste. This study compared hydrogen production resulting from fermentation using raw and detoxified tequila vinasse. Vinasse was detoxified with granular activated charcoal, which was used to adsorb compounds that could inhibit the production of hydrogen by dark fermentation. In batch cultures detoxification of vinasse led to up to 20% higher maximum velocities of hydrogen production, a 5.4 h reduction in the lag phase and an 11% higher molar yield, compared to results obtained with raw vinasse. Losses of sugars after detoxification provoked that the specific hydrogen volumetric yields obtained with detoxified vinasse were 30–40% lower with 5 g COD/L and 15 g COD/L initial concentrations, compared to the ones obtained with raw vinasse. For an initial 30 g COD/L no differences in specific hydrogen yields were observed between raw or detoxified vinasse in batch fermentation. Continuous culture fermentation of vinasse showed hydrogen production rates between 1.32 ± 0.07 to 1.39 ± 0.14 NL H2/L-d when extra nutrients were added, while a stable production of hydrogen through fermentation of detoxified vinasse could not be maintained despite nutrient addition. Production of hydrogen from vinasse diluted with water with no additional nutrients was assessed and rates close to 0.42 ± 0.02 NL H2/L-d and hydrogen content close to 37% were obtained. Accumulation of lactic acid and a predominant production of butyric acid over acetic acid suggested that the fermentation dynamics of vinasse with no supplementary nutrients were especially susceptible to high substrate loading rates and prolonged hydraulic retention times.

Efficient Synthesis of Methyl 3-Acetoxypropionate by a Newly Identified Baeyer-Villiger Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are an emerging class of promising biocatalysts for the oxidation of ketones to prepare corresponding esters or lactones. Although many BVMOs have been reported, the development of highly efficient enzymes for use in industrial applications is desirable. In this work, we identified a BVMO from Rhodococcus pyridinivorans (BVMORp) with a high affinity toward aliphatic methyl ketones (Km < 3.0 μM). The enzyme was highly soluble and relatively stable, with a half-life of 23 h at 30°C and pH 7.5. The most effective substrate discovered so far is 2-hexanone (kcat = 2.1 s-1; Km = 1.5 μM). Furthermore, BVMORp exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMORp as the catalyst, a high concentration (26.0 g/liter; 200 mM) of methyl levulinate was completely converted to methyl 3-acetoxypropionate after 4 h, with a space-time yield of 5.4 g liter-1 h-1 Thus, BVMORp is a promising biocatalyst for the synthesis of 3-hydroxypropionate from readily available biobased levulinate to replace the conventional fermentation.IMPORTANCE BVMOs are emerging as a green alternative to traditional oxidants in the BV oxidation of ketones. Although many BVMOs are discovered and used in organic synthesis, few are really applied in industry, especially in the case of aliphatic ketones. Herein, a highly soluble and relatively stable monooxygenase from Rhodococcus pyridinivorans (BVMORp) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMORp possesses unusually high substrate loading during the catalysis of the oxidation of biobased methyl levulinate to 3-hydroxypropionic acid derivatives. This study indicates that the synthesis of 3-hydroxypropionate from readily available biobased levulinate by BVMORp-catalyzed oxidation holds great promise to replace traditional fermentation.

Flavin Oxidoreductase‐Mediated Regeneration of Nicotinamide Adenine Dinucleotide with Dioxygen and Catalytic Amount of Flavin Mononucleotide for One‐Pot Multi‐Enzymatic Preparation of Ursodeoxycholic Acid

AbstractUrsodeoxycholic acid (UDCA), a pharmaceutical ingredient widely used in clinics, can be prepared from chenodeoxycholic acid (CDCA) by the epimerization of the 7α‐OH group. In this study, a nicotinamide adenine dinucleotide (NAD+) regeneration system was developed by using flavin oxidoreductase (FR) and flavin mononucleotide (FMN). Only catalytic amount of FMN is required for the effective NAD+ recycling. FR/FMN system was then applied in the oxidation of CDCA to 7‐ketolithocholic acid (7‐keto‐LCA) by NAD+‐dependent 7α‐hydroxysteroid dehydrogenase (Bs‐7α‐HSDH) from Brevundimonas sp., which showed extremely high enzyme activity toward CDCA (kcat/Km=8050 s−1 ⋅ mM−1). When Escherichia coli whole cells coexpressing Bs‐7α‐HSDH and FR genes were used as biocatalyst, CDCA (50 mM) was completely converted to 7‐keto‐LCA with the turnover number of FMN being 227 and 58.8 g ⋅ L−1 ⋅ d−1 space‐time yield of 7‐keto‐LCA. For the reduction of 7‐keto‐LCA, nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent 7‐β‐hydroxysteroid dehydrogenase (Cm‐7β‐HSDH) from Clostridium sp. Marseille was employed with alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH) and iso‐propanol as co‐factor regeneration system. When E. coli whole cells coexpressing Cm‐7β‐HSDH and TbADH genes were used as biocatalyst, 40 mM 7‐keto‐LCA was reduced to UDCA with 26.8 g ⋅ L−1 ⋅ d−1 space‐time yield. The oxidation and reduction were then carried in a one‐pot concurrent mode, 12.5 mM CDCA was completely converted to UDCA. The epimerization of CDCA to UDCA proceeded to completion at the substrate concentration of 30 mM in the one‐pot sequential process. Therefore, the complete conversion of CDCA to UDCA in one‐pot has been realized by employing 7α‐HSDH and 7β‐HSDH of different co‐factor specificities with independent co‐factor recycling systems. The cholic acids, especially UDCA, exert inhibitive effect on the activities of these enzymes, preventing the complete epimerization of 7α‐OH at higher substrate loading. This inhibition issue should be solvable by engineering the involved enzymes, that is currently pursued in our laboratory.magnified image

Enzymatic Synthesis of Aliphatic Nitriles at a Substrate Loading of up to 1.4 kg/L: A Biocatalytic Record Achieved with a Heme Protein.

A biocatalytic approach toward linear aliphatic nitriles being widely used as industrial bulk chemicals has been developed that runs at high substrate loadings of up to 1.4 kg/L as demonstrated for the synthesis of n-octanenitrile. This substrate loading is one of the highest ever reported in biocatalysis and to best of our knowledge the highest obtained for a water-immiscible product in aqueous medium. It is noteworthy that the biotransformation at such a high substrate loading was achieved by means of a metalloprotein bearing an iron-containing heme subunit in the active site. In detail, an aldoxime dehydratase from Bacillus sp. OxB-1 was used as a biocatalyst for a dehydration of aldoximes as readily available starting materials due to their easy preparation from aliphatic aldehydes through spontaneous condensation with hydroxylamine as bulk chemical. Excellent conversions toward the nitriles in the two-phase system were achieved and the products are easily separated from the reaction mixture without the need for further purification. Aliphatic nitriles are used in industry as solvents and intermediates for the production of surfactants and life sciences products.

Kinetic and molecular dynamics study of inhibition and transglycosylation in Hypocrea jecorina family 3 β-glucosidases

β-Glucosidases enhance enzymatic biomass conversion by relieving cellobiose inhibition of endoglucanases and cellobiohydrolases. However, the susceptibility of these enzymes to inhibition and transglycosylation at high glucose or cellobiose concentrations severely limits their activity and, consequently, the overall efficiency of enzyme mixtures. We determined the impact of these two processes on the hydrolytic activity of the industrially relevant family 3 β-glucosidases from Hypocrea jecorina, HjCel3A and HjCel3B, and investigated the underlying molecular mechanisms through kinetic studies, binding free energy calculations, and molecular dynamics (MD) simulations. HjCel3B had a 7-fold higher specificity for cellobiose than HjCel3A but greater tendency for glucose inhibition. Energy decomposition analysis indicated that cellobiose has relatively weak electrostatic interactions with binding site residues, allowing it to be easily displaced by glucose and free to inhibit other hydrolytic enzymes. HjCel3A is, thus, preferable as an industrial β-glucosidase despite its lower activity caused by transglycosylation. This competing pathway to hydrolysis arises from binding of glucose or cellobiose at the product site after formation of the glycosyl-enzyme intermediate. MD simulations revealed that binding is facilitated by hydrophobic interactions with Trp-37, Phe-260, and Tyr-443. Targeting these aromatic residues for mutation to reduce substrate affinity at the product site would therefore potentially mitigate transglycosidic activity. Engineering improved variants of HjCel3A and other structurally similar β-glucosidases would have a significant economic effect on enzymatic biomass conversion in terms of yield and production cost as the process can be consequently conducted at higher substrate loadings.

Development of an Efficient and Cost-Effective Enzymatic Process for Production of (R)-[3,5-bis(trifluoromethyl)phenyl] Ethanol Using Carbonyl Reductase Derived from Leifsonia sp. S749.

(R)-[3,5-bis(trifluoromethyl) phenyl] ethanol [(R)-3,5-BTPE] is a crucial chiral intermediate for the synthesis of the NK-1 receptor antagonists aprepitant, rolapitant and fosaprepitant. The carbonyl reductase KR01 from Leifsonia sp. S749, discovered by protein sequence alignment, could convert 3',5'-bis(trifluoromethyl) acetophenone (3,5-BTAP) into (R)-3,5-BTPE with excellent activity and enantioselectivity. In order to enhance the conversion efficiency at high substrate concentrations, the reaction conditions were optimized by response surface analysis. The results showed that 600g/L 3,5-BTAP was bioreduced to (R)-3,5-BTPE (> 99.9% enantiomeric excess) by the recombinant Escherichia coli/pET-28a (+)-KR01 whole cells, with a 98.3% conversion and 59g/L/h productivity under the optimized reaction conditions. In addition, the recombinant E. coli cells could be repeatedly used up to seven times in the reaction mixture containing 90% isopropanol (IPA). This is the highest substrate loading and productivity for the bioreduction of 3,5-BTAP by carbonyl reductase ever reported, and this method represents an efficient and cost-effective process for production of (R)-3,5-BTPE.

Application of an immobilized Rhizopus oryzae lipase to batch and continuous ester synthesis with a mixture of a lauric acid and fusel oil

Five commercial lipase preparations immobilized on epoxy-polysiloxane- hydroxyethylcellulose were tested as potential biocatalysts to mediate the esterification reaction of isoamyl alcohol with lauric acid. Among them, the immobilized lipase from Rhizopus oryzae was the most active biocatalyst, attained high ester yield (81.26%) in 24 h, being selected for subsequent tests using fusel oil as a source of isoamyl alcohol under batch and continuous runs. Under batch conditions, no adverse effect was found when pure isoamyl alcohol was replaced by fusel oil. However, under continuous runs, the selected biocatalyst was found to have limited performance (half-life time = 173 h) and the average ester productivity was only 277.8 ± 19.3 μmol g−1 min−1. These results can be credited to the high affinity of the hybrid matrix for the water formed as a by-product, modifying the substrate interaction with the essential water around the enzyme limiting the substrate migration to the solid phase and thereby reducing the enzyme activity. By replacing the hybrid support for a higher hydrophobic matrix such as commercial copolymer styrene-divinylbenzene it was possible to attain a stable operation, achieving an average ester productivity of 794.43 ± 66.04 μmol g−1 min−1 and increasing the half-life time to 285 h. Further productivity incremental (1598.68 ± 135.79 μmol g−1 min−1) was also attained by using higher substrate loading. This process shows to be an attractive alternative to bulk chemical routes to satisfy increasing commercial demands for isoamyl laurate an essential emollient in the cosmetic industry.

Improvement of waste-fed bioelectrochemical system performance by selected electro-active microbes: Process evaluation and a kinetic study

In this work, bioaugmentation strategy was tested to enhance electricity production efficiency from municipal waste liquor feedstock in microbial fuel cells (MFC). During the experiments, MFCs inoculated with a mixed anaerobic consortium were enriched by several pure, electro-active bacterial cultures (such as Propionibacterium freudenreichii, Cupriavidus basilensis and Lactococcus lactis) and behaviours were assessed kinetically. It turned out that energy yield could be enhanced mainly at high substrate loadings. Furthermore, energy production and COD removal rate showed an optimum and could be characterized by a saturation range within the applied COD loadings, which could be elucidated applying the Monod-model for describing intracellular losses. Polarization measurements showed the positive effect of bioaugmentation also on extracellular losses. The data indicated a successful augmentation process for enhancing MFC efficiency, which was utmost in case of augmentation strain of Propionibacterium freudenreichii.

Impact of blending on hydrolysis and ethanol fermentation of garden wastes

Abstract Mixed biomass has the characteristics of stable supply and low cost of collection, and its bio-refinery could provide an effective contribution to the sustainable development of fuel ethanol production. Compared to traditional single energy crops, the key challenge of mixed biomass conversion lies in overcoming the non-uniformity of blended materials. In this study, the influence of blending on hydrolysis and ethanol fermentation of garden wastes was investigated. There was an unexpected result in that the total xylose yield during a liquid hot water process was enhanced when different garden biomasses were mixed. The peak of total xylose recovery appeared at the value of ∼0.5 for the ratio of xylan to lignin, which was further confirmed by blending multiple biomasses. Moreover, the non-uniformity of physio-chemical characteristics in mixed garden wastes restricted the co-hydrolysis of cellulose, particularly in the mixed-five-biomass. In spite of this disadvantage, blended feedstocks showed a better flow behavior, which could make them more suitable for enzymatic hydrolysis and ethanol fermentation with high substrate loading. The 72 h enzymatic digestibility and glucose concentration of treated mixed-three-biomass reached 100 ± 5% and 52.3 ± 2.6 g/l under the conditions of 15% (w/v) substrate loading, with the addition of 1% (v/v) Tween 20, which gave a maximum ethanol concentration of 16.9 ± 0.8 g/l. Overall, these results have shown that the complementary or synergistic effects of different individual biomasses make it feasible to produce ethanol from mixed biomass.

Methanosarcina plays a main role during methanogenesis of high-solids food waste and cardboard

Anaerobic digestion of food waste is a complex process often hindered by high concentrations of volatile fatty acids and ammonia. Methanogenic archaea are more sensitive to these inhibitors than bacteria and thus the structure of their community is critical to avoid reactor acidification. In this study, the performances of three different inocula were compared using batch digestion tests of food waste and cardboard mixtures. Particular attention was paid to the archaeal communities in the inocula and after digestion. While the tests started with inocula rich in Methanosarcina led to efficient methane production, VFAs accumulated in the reactors where inocula initially were poor in this archaea and no methane was produced. In addition, higher substrate loads were tolerated when greater proportions of Methanosarcina were initially present in the inoculum. Independently of the inoculum origin, Methanosarcina were the dominant methanogens in the digestates from the experiments that efficiently produced methane. These results suggest that the initial archaeal composition of the inoculum is crucial during reactor start-up to achieve stable anaerobic digestion at high concentrations of ammonia and organic acids.

Highly efficient production of 1-cyanocyclohexaneacetic acid by cross-linked cell aggregates (CLCAs) of recombinant E. coli harboring nitrilase gene

Abstract 1-Cyanocyclohexaneacetic acid (1-CHAA) is an important intermediate in the preparation of gabapentin. Regioselective biotransformation of 1-cyanocyclohexaneacetonitile (1-CHAN) into 1-CHAA, mediated by the immobilization of recombinant microbial nitrilase, is a novel and promising strategy for its production. In this study, a recombinant E. coli expressing Acidovorax facilis nitrilase was flocculated with polyethylenemine, followed by cross-linking with glutaraldehyde to obtain cross-linked cell aggregates (CLCAs). Under the optimum preparation conditions, the activity recovery of CLCAs reached 68%. Characterization of the CLCAs showed that the optimum pH was not affected by cross-linking, but cross-linking resulted in slightly different optimum temperature. The half-life of CLCAs at 45 °C was 266.5 h, which is 11.6-times higher than that of the free cells. Preparative-scale biotransformation of 1-CHAN to 1-CHAA at high substrate loading (up to 1.0 M) was performed using CLCAs as the biocatalyst in a 2L stirred reactor, resulting in an conversion of 91%, with a productivity of 244.5 g L−1 d−1. The CLCAs also exhibited a high operational stability and even after 30 cycles of reaction, it retained >80% of the initial conversion.