Microbial alchemy: upcycling of brewery spent grains into high-value products through fermentation.

Spent grains are one of the lignocellulosic biomasses available in abundance, discarded by breweries as waste. The brewing process generates around 25-30% of waste in different forms and spent grains alone account for 80-85% of that waste, resulting in a significant global waste volume. Despite containing essential nutrients, i.e., carbohydrates, fibers, proteins, fatty acids, lipids, minerals, and vitamins, efficient and economically viable valorization of these grains is lacking. Microbial fermentation enables the valorization of spent grain biomass into numerous commercially valuable products used in energy, food, healthcare, and biomaterials. However, the process still needs more investigation to overcome challenges, such as transportation, cost-effective pretreatment, and fermentation strategy. to lower the product cost and to achieve market feasibility and customer affordability. This review summarizes the potential of spent grains valorization via microbial fermentation and associated challenges.

Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay.

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Evaluation of simplified ester-linked fatty acid analysis (ELFA) for phospholipid fatty acid (PLFA) analysis of bacterial population.

Phospholipid fatty acid (PLFA) analysis is used for characterizing microbial communities based on their lipid profiles. This method avoids biases from PCR or culture, allowing data collection in a natural state. However, PLFA is labor-intensive due to lipid fractionation. Simplified ester-linked fatty acid analysis (ELFA), which skips lipid fractionation, offers an alternative. It utilizes base-catalyzed methylation to derivatize only lipids, not free fatty acids, and found glycolipid and neutral lipid fractions are scarcely present in most bacteria, allowing lipid fractionation to be skipped. ELFA method showed a high correlation to PLFA data (r = 0.99) and higher sensitivity than the PLFA method by 1.5-2.57-fold, mainly due to the higher recovery of lipids, which was 1.5-1.9 times higher than with PLFA. The theoretical limit of detection (LOD) and limit of quantification (LOQ) for the ELFA method indicated that 1.54-fold less sample was needed for analysis than with the PLFA method. Our analysis of three bacterial cultures and a simulated consortium revealed the effectiveness of the ELFA method by its simple procedure and enhanced sensitivity for detecting strain-specific markers, which were not detected in PLFA analysis. Overall, this method could be easily used for the population analysis of synthetic consortia.

A review on microbes mediated resource recovery and bioplastic (polyhydroxyalkanoates) production from wastewater.

BACKGROUND: Plastic is widely utilized in packaging, frameworks, and as coverings material. Its overconsumption and slow degradation, pose threats to ecosystems due to its toxic effects. While polyhydroxyalkanoates (PHA) offer a sustainable alternative to petroleum-based plastics, their production costs present significant obstacles to global adoption. On the other side, a multitude of household and industrial activities generate substantial volumes of wastewater containing both organic and inorganic contaminants. This not only poses a threat to ecosystems but also presents opportunities to get benefits from the circular economy. Production of bioplastics may be improved by using the nutrients and minerals in wastewater as a feedstock for microbial fermentation. Strategies like feast-famine culture, mixed-consortia culture, and integrated processes have been developed for PHA production from highly polluted wastewater with high organic loads. Various process parameters like organic loading rate, organic content (volatile fatty acids), dissolved oxygen, operating pH, and temperature also have critical roles in PHA accumulation in microbial biomass. Research advances are also going on in downstream and recovery of PHA utilizing a combination of physical and chemical (halogenated solvents, surfactants, green solvents) methods. This review highlights recent developments in upcycling wastewater resources into PHA, encompassing various production strategies, downstream processing methodologies, and techno-economic analyses. SHORT CONCLUSION: Organic carbon and nitrogen present in wastewater offer a promising, cost-effective source for producing bioplastic. Previous attempts have focused on enhancing productivity through optimizing culture systems and growth conditions. However, despite technological progress, significant challenges persist, such as low productivity, intricate downstream processing, scalability issues, and the properties of resulting PHA.

Novel differential scanning calorimetry (DSC) application to select polyhydroxyalkanoate (PHA) producers correlating 3-hydroxyhexanoate (3-HHx) monomer with melting enthalpy.

Polyhydroxyalkanoate (PHA) is an environmental alternative to petroleum-based plastics because of its biodegradability. The polymer properties of PHA have been improved by the incorporation of different monomers. Traditionally, the monomer composition of PHA has been analyzed using gas chromatography (GC) and nuclear magnetic resonance (NMR), providing accurate monomer composition. However, sequential analysis of the thermal properties of PHA using differential scanning calorimetry (DSC) remains necessary, providing crucial insights into its thermal characteristics. To shorten the monomer composition and thermal property analysis, we directly applied DSC to the analysis of the obtained PHA film and observed a high correlation (r2 = 0.98) between melting enthalpy and the 3-hydroxyhexanoate (3-HHx) mole fraction in the polymer. A higher 3-HHx fraction resulted in a lower melting enthalpy as 3-HHx provided the polymer with higher flexibility. Based on this, we selected the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) producing strain from Cupriavidus strains that newly screened and transformed with vectors containing P(3HB-co-3HHx) biosynthetic genes, achieving an average error rate below 1.8% between GC and DSC results. Cupriavidus sp. BK2 showed a high 3-HHx mole fraction, up to 10.38 mol%, with Tm (℃) = 171.5 and ΔH of Tm (J/g) = 48.0, simultaneously detected via DSC. This study is an example of the expansion of DSC for PHA analysis from polymer science to microbial engineering.

Indigo production goes green: a review on opportunities and challenges of fermentative production.

Indigo is a widely used dye in various industries, such as textile, cosmetics, and food. However, traditional methods of indigo extraction and processing are associated with environmental and economic challenges. Fermentative production of indigo using microbial strains has emerged as a promising alternative that offers sustainability and cost-effectiveness. This review article provides a critical overview of microbial diversity, metabolic pathways, fermentation strategies, and genetic engineering approaches for fermentative indigo production. The advantages and limitations of different indigo production systems and a critique of the current understanding of indigo biosynthesis are discussed. Finally, the potential application of indigo in other sectors is also discussed. Overall, fermentative production of indigo offers a sustainable and bio-based alternative to synthetic methods and has the potential to contribute to the development of sustainable and circular biomanufacturing.

Optimization of polyhydroxyalkanoate production in Halomonas sp. YLGW01 using mixed volatile fatty acids: a study on mixture analysis and fed-batch strategy.

Polyhydroxyalkanoate (PHA) is one of the most promising materials for replacing petroleum-based plastics, and it can be produced from various renewable biomass sources. In this study, PHA production was conducted using Halomonas sp. YLGW01 utilizing mixed volatile fatty acids (VFAs) as carbon sources. The ratio and concentration of carbon and nitrogen sources were optimized through mixture analysis and organic nitrogen source screening, respectively. It was found that the highest cell dry weight (CDW) of 3.15 g/L and PHA production of 1.63 g/L were achieved when the ratio of acetate to lactate in the mixed VFAs was 0.45:0.55. Furthermore, supplementation of organic nitrogen sources such as soytone resulted in a ninefold increase in CDW (reaching 2.32 g/L) and a 22-fold increase in PHA production (reaching 1.60 g/L) compared to using inorganic nitrogen sources. Subsequently, DO-stat, VFAs consumption rate stat, and pH-stat fed-batch methods were applied to investigate and evaluate PHA productivity. The results showed that when pH-stat-based VFAs feeding was employed, a CDW of 7 g/L and PHA production of 5.1 g/L were achieved within 68 h, with a PHA content of 73%. Overall, the pH-stat fed-batch strategy proved to be effective in enhancing PHA production by Halomonas sp. YLGW01 utilizing VFAs.

A strategy to promote the convenient storage and direct use of polyhydroxybutyrate-degrading Bacillus sp. JY14 by lyophilization with protective reagents.

BACKGROUND: Bioplastics are attracting considerable attention, owing to the increase in non-degradable waste. Using microorganisms to degrade bioplastics is a promising strategy for reducing non-degradable plastic waste. However, maintaining bacterial viability and activity during culture and storage remains challenging. With the use of conventional methods, cell viability and activity was lost; therefore, these conditions need to be optimized for the practical application of microorganisms in bioplastic degradation. Therefore, we aimed to optimize the feasibility of the lyophilization method for convenient storage and direct use. In addition, we incoporated protective reagents to increase the viability and activity of lyophilized microorganisms. By selecting and applying the best protective reagents for the lyophilization process and the effects of additives on the growth and PHB-degrading activity of strains were analyzed after lyophilization. For developing the lyophilization method for protecting degradation activity, it may promote practical applications of bioplastic-degrading bacteria. RESULTS: In this study, the polyhydroxybutyrate (PHB)-degrading strain, Bacillus sp. JY14 was lyophilized with the use of various sugars as protective reagents. Among the carbon sources tested, raffinose was associated with the highest cell survival rate (12.1%). Moreover, 7% of raffionose showed the highest PHB degradation yield (92.1%). Therefore, raffinose was selected as the most effective protective reagent. Also, bacterial activity was successfully maintained, with raffinose, under different storage temperatures and period. CONCLUSIONS: This study highlights lyophilization as an efficient microorganism storage method to enhance the applicability of bioplastic-degrading bacterial strains. The approach developed herein can be further studied and used to promote the application of microorganisms in bioplastic degradation.

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters.

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, ketoacids, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including ketoisovalerate submodule for converting pyruvate to ketoisovalerate, ketoacid elongation submodule for producing longer carbon-chain ketoacids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23-31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (>0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

Simultaneous monitoring of each component on degradation of blended bioplastic using gas chromatography-mass spectrometry.

The increasing interest in bioplastics, with regard to future environmental issues, has rendered research on bioplastic biodegradation highly important. However, only a few tools directly monitor the degradation of bioplastics without measuring the levels of gaseous products, such as carbon dioxide. Classical nonquantitative methods, such as clear zone tests on solid plates, and less-sensitive weight-loss experiments in liquid media measured using a precision scale, are still employed to screen the microbial players associated with bioplastic degradation and monitor the biodegradation rates. However, the simultaneous monitoring of the degradation of each component of blended bioplastics has not been previously reported. In the present study, to provide information regarding the degradation rates and compositional changes of different bioplastics in a blend in a time-dependent manner, we simultaneously monitored and quantified the degradation of four bioplastics, polyhydroxybutyrate (PHB), polybutylene succinate (PBS), polycaprolactone (PCL), and poly(butylene adipate-co-terephthalate) (PBAT), by Bacillus sp. JY36 using gas chromatography-mass spectrometry (GC-MS) analysis after fatty acid methyl ester (FAME) derivatization. Our results demonstrate the feasibility of using the GC-MS-based method described here to obtain comprehensive data regarding blended bioplastics and their degradation. Moreover, our findings indicate that this method may support classical analytic tools for assessing bioplastic biodegradation.

Constructing multi-enzymatic cascade reactions for selective production of 6-bromoindirubin from tryptophan in Escherichia coli.

6-Bromoindirubin (6BrIR), found in Murex sea snails, is a precursor of indirubin-derivatives anticancer drugs. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and difficulties in site-specific bromination and oxidation at the indole ring. Here, we present an efficient 6BrIR production strategy in Escherichia coli by using four enzymes, that is, tryptophan 6-halogenase fused with flavin reductase Fre (Fre-L3-SttH), tryptophanase (TnaA), toluene 4-monooxygenase (PmT4MO), and flavin-containing monooxygenase (MaFMO). Although most indole oxygenases preferentially oxygenate the electronically active C3 position of indole, PmT4MO was newly characterized to perform C2 oxygenation of 6-bromoindole with 45% yield to produce 6-bromo-2-oxindole. In addition, 6BrIR was selectively generated without indigo and indirubin byproducts by controlling the reducing power of cysteine and oxygen supply during the MaFMO reaction. These approaches led to 34.1 mg/L 6BrIR productions, making it possible to produce the critical precursor of the anticancer drugs only from natural ingredients such as tryptophan, NaBr, and oxygen.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Microbial synthesis of violacein pigment and its potential applications.

Violacein is a pigment synthesized by Gram-negative bacteria such as Chromobacterium violaceum. It has garnered significant interest owing to its unique physiological and biological activities along with its synergistic effects with various antibiotics. In addition to C. violaceum, several microorganisms, including: Duganella sp., Pseudoalteromonas sp., Iodobacter sp., and Massilia sp., are known to produce violacein. Along with the identification of violacein-producing strains, the genetic regulation, quorum sensing mechanism, and sequence of the vio-operon involved in the biosynthesis of violacein have been elucidated. From an engineering perspective, the heterologous production of violacein using the genetically engineered Escherichia coli or Citrobacter freundii host has also been attempted. Genetic engineering of host cells involves the heterologous expression of genes involved in the vio operon and the optimization of metabolic pathways and gene regulation. Further, the crystallography of VioD and VioE was revealed, and mass production by enzyme engineering has been accelerated. In this review, we highlight the biologically assisted end-use applications of violacein (such as functional fabric development, nanoparticles, functional polymer composites, and sunscreen ingredients) and violacein activation mechanisms, production strains, and the results of mass production with engineered methods. The prospects for violacein research and engineering applications have also been discussed.

Bacillus subtilis as a robust host for biochemical production utilizing biomass.

Bacillus subtilis is regarded as a suitable host for biochemical production owing to its excellent growth and bioresource utilization characteristics. In addition, the distinct endogenous metabolic pathways and the suitability of the heterologous pathways have made B. subtilis a robust and promising host for producing biochemicals, such as: bioalcohols; bioorganic acids (lactic acids, α-ketoglutaric acid, and γ-aminobutyric acid); biopolymers (poly(γ-glutamic acid, polyhydroxyalkanoates (PHA), and polysaccharides and monosaccharides (N-acetylglucosamine, xylooligosaccharides, and hyaluronic acid)); and bioflocculants. Also for producing oligopeptides and functional peptides, owing to its efficient protein secretion system. Several metabolic and genetic engineering techniques, such as target gene overexpression and inactivation of bypass pathways, have led to the improvement in production titers and product selectivity. In this review article, recent progress in the utilization of robust B. subtilis-based host systems for biomass conversion and biochemical production has been highlighted, and the prospects of such host systems are suggested.

Perturbation of the peptidoglycan network and utilization of the signal recognition particle-dependent pathway enhances the extracellular production of a truncational mutant of CelA in Escherichia coli.

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.

6,8-Diprenylorobol induces apoptosis in human colon cancer cells via activation of intracellular reactive oxygen species and p53.

6,8-Diprenylorobol is a natural compound mainly found in Glycyrrhiza uralensis fisch and Maclura tricuspidata, which has been used traditionally as food and medicine in Asia. So far, the antiproliferative effect of 6,8-diprenylorobol has not been studied yet in colon cancer. In this study, we aimed to evaluate the antiproliferative effects of 6,8-diprenylorobol in LoVo and HCT15, two kinds of human colon cancer cells. 6,8-Diprenylorobol inhibited the proliferation of LoVo and HCT15 cells in a dose- and time-dependent manner. A 40 µM of 6,8-diprenylorobol for 72 h reduced both of cell viability under 50%. After treatment of 6,8-diprenylorobol (40 and 60 µM) for 72 h, late apoptotic cell portion in LoVo and HCT15 cells were 24, 70% and 13, 90%, respectively, which was confirmed by checking DNA fragmentation in both cells. Mechanistically, 6,8-diprenylorobol activated p53 and its phosphorylated form (Ser15, Ser20, and Ser46) expression but suppressed Akt and mitogen-activated protein kinases (MAPKs) phosphorylation in LoVo and HCT15 cells. Interestingly, 6,8-diprenylorobol induced the generation of intracellular reactive oxygen species (ROS), which was attenuated with N-acetyl cysteine (NAC) treatment. Compared to the control, 60 µM of 6,8-diprenylorobol caused to increase ROS level to 210% in LoVo and HCT15, which was reduced into 161% and 124%, respectively with NAC. Furthermore, cell viability and apoptotic cell portion by 6,8-diprenylorobol was recovered by incubation with NAC. Taken together, these results indicate that 6,8-diprenylorobol has the potential antiproliferative effect against LoVo and HCT15 colon cancer cells through activation of p53 and generation of ROS.

Improvement of cadaverine production in whole cell system with baker's yeast for cofactor regeneration.

Cadaverine, 1,5-diaminopentane, is one of the most promising chemicals for biobased-polyamide production and it has been successfully produced up to molar concentration. Pyridoxal 5'-phosphate (PLP) is a critical cofactor for inducible lysine decarboxylase (CadA) and is required up to micromolar concentration level. Previously the regeneration of PLP in cadaverine bioconversion has been studied and salvage pathway pyridoxal kinase (PdxY) was successfully introduced; however, this system also required a continuous supply of adenosine 5'-triphosphate (ATP) for PLP regeneration from pyridoxal (PL) which add in cost. Herein, to improve the process further a method of ATP regeneration was established by applying baker's yeast with jhAY strain harboring CadA and PdxY, and demonstrated that providing a moderate amount of adenosine 5'-triphosphate (ATP) with the simple addition of baker's yeast could increase cadaverine production dramatically. After optimization of reaction conditions, such as PL, adenosine 5'-diphosphate, MgCl2, and phosphate buffer, we able to achieve high production (1740 mM, 87% yield) from 2 M L-lysine. Moreover, this approach could give averaged 80.4% of cadaverine yield after three times reactions with baker's yeast and jhAY strain. It is expected that baker's yeast could be applied to other reactions requiring an ATP regeneration system.

Sorption of brilliant green dye using soybean straw-derived biochar: characterization, kinetics, thermodynamics and toxicity studies.

The present study was aimed to investigate brilliant green (BG) dye sorption onto soybean straw biochar (SSB) prepared at 800 °C and further understanding the sorption mechanism. Sorption kinetic models such as pseudo-first and pseudo-second order were executed for demonstrating sorption mechanism between the dye and biochar. Results of kinetics study were fitted well to pseudo-second-order kinetic model (R2 0.997) indicating that the reaction followed chemisorption mechanism. Furthermore, the effect of various parameters like sorbent dose, dye concentration, incubation time, pH and temperature on dye sorption was also studied. The maximum dye removal percentage and sorption capacity for SSB (800 °C) within 60 min were found to be 99.73% and 73.50 mg g- 1, respectively, at pH 8 and 60 °C temperature, whereas adsorption isotherm studies showed a higher correlation coefficient values for Freundlich model (R2 0.990-0.996) followed by Langmuir model suggesting that sorption process was multilayer. The characterization of biomass and biochar was performed with the aid of analytical techniques like scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), Brunauer-Emmett-Teller (BET) theory, X-ray diffraction (XRD) and thermo-gravimetric analysis (TGA). FTIR analysis showed active groups on biochar surface. BET study revealed higher surface area of biochar (194.7 m2/g) than the biomass (12.84 m2/g). Besides, phyto- and cytogenotoxic studies revealed significant decrease in the toxicity of dye containing water after treating with SSB. Therefore, this study has proved the sorption potential of soybean straw biochar for BG dye and could be further considered as sustainable cost-effective strategy for treating the textile dye-contaminated wastewater.

Simultaneous monitoring of the bioconversion from lysine to glutaric acid by ethyl chloroformate derivatization and gas chromatography-mass spectrometry.

Glutaric acid is a precursor of a plasticizer that can be used for the production of polyester amides, ester plasticizer, corrosion inhibitor, and others. Glutaric acid can be produced either via bioconversion or chemical synthesis, and some metabolites and intermediates are produced during the reaction. To ensure reaction efficiency, the substrates, intermediates, and products, especially in the bioconversion system, should be closely monitored. Until now, high performance liquid chromatography (HPLC) has generally been used to analyze the glutaric acid-related metabolites, although it demands separate time-consuming derivatization and non-derivatization analyses. To substitute for this unreasonable analytical method, we applied herein a gas chromatography - mass spectrometry (GC-MS) method with ethyl chloroformate (ECF) derivatization to simultaneously monitor the major metabolites. We determined the suitability of GC-MS analysis using defined concentrations of six metabolites (l-lysine, cadaverine, 5-aminovaleric acid, 2-oxoglutaric acid, glutamate, and glutaric acid) and their mass chromatograms, regression equations, regression coefficient values (R2), dynamic ranges (mM), and retention times (RT). This method successfully monitored the production process in complex fermentation broth.

Effects of a Δ-9-fatty acid desaturase and a cyclopropane-fatty acid synthase from the novel psychrophile Pseudomonas sp. B14-6 on bacterial membrane properties.

Psychrophilic bacteria, living at low and mild temperatures, can contribute significantly to our understanding of microbial responses to temperature, markedly occurring in the bacterial membrane. Here, a newly isolated strain, Pseudomonas sp. B14-6, was found to dynamically change its unsaturated fatty acid and cyclic fatty acid content depending on temperature which was revealed by phospholipid fatty acid (PLFA) analysis. Genome sequencing yielded the sequences of the genes Δ-9-fatty acid desaturase (desA) and cyclopropane-fatty acid-acyl-phospholipid synthase (cfa). Overexpression of desA in Escherichia coli led to an increase in the levels of unsaturated fatty acids, resulting in decreased membrane hydrophobicity and increased fluidity. Cfa proteins from different species were all found to promote bacterial growth, despite their sequence diversity. In conclusion, PLFA analysis and genome sequencing unraveled the temperature-related behavior of Pseudomonas sp. B14-6 and the functions of two membrane-related enzymes. Our results shed new light on temperature-dependent microbial behaviors and might allow to predict the consequences of global warming on microbial communities.