Application of quantitative IR spectral analysis of bacterial cells to acetone-butanol-ethanol fermentation monitoring

Clostridium acetobutylicum ATCC 824 was grown under three different acetone-butanol-ethanol (ABE) fermentation conditions: (1) strictly anaerobic conditions with vegetative inoculum; (2) semi-anaerobic conditions with vegetative inoculum; and (3) strictly anaerobic conditions with spore inoculum. Semi-anaerobic fermentation with vegetative inoculum and strictly anaerobic fermentation with spore inoculum produced solvents at high level. Strictly anaerobic fermentation with vegetative inoculum showed an “acid crash”, i.e. produced mainly acids and did not switch to predominant solvent production. The content of carbohydrates, nucleic acids, proteins and lipids in Clostridium cells during the fermentation were evaluated from the mid-IR spectra. The content of nucleic acids decreased with process time, and the lipid content increased, corresponding to ceasing growth and formation of the toxic fermentation products. It was shown that the physiological states of either solvent production or acid crash are reflected in the microbial biomass composition, which can be assessed by IR spectroscopy.     

Elimination of Acetate Production to Improve Ethanol Yield During Continuous Synthesis Gas Fermentation by Engineered Biocatalyst Clostridium sp. MTEtOH550

Acetogen strain Clostridum sp. MT653 produced acetate 273?mM (p?<?0.005) and ethanol 250?mM (p?<?0.005) from synthesis gas blend mixture of 64?% CO and 36?%?H₂. Clostridum sp. MT653 was metabolically engineered to the biocatalyst strain Clostridium sp. MTEtOH550. The biocatalyst increased ethanol yield to 590?mM with no acetate production during single-stage continuous syngas fermentation due to expression of synthetic adh cloned in a multi-copy number expression vector. The acetate production was eliminated by inactivation of the pta gene in Clostridium sp. MTEtOH550. Gene introduction and gene elimination were achieved only using Syngas Biofuels Energy, Inc. electroporation generator. The electrotransformation efficiencies were 8.0?±?0.2?×?10⁶ per microgram of transforming DNA of the expression vector at cell viability ~15?%. The frequency of suicidal vector integration to inactivate pta was ~10^?5 per the number of recipient cells. This is the first report on elimination of acetate production and overexpression of synthetic adh gene to engineer acetogen biocatalyst for selective biofuel ethanol production during continuous syngas fermentation.     

Cre-lox66/lox71-Based Elimination of Phosphotransacetylase or Acetaldehyde Dehydrogenase Shifted Carbon Flux in Acetogen Rendering Selective Overproduction of Ethanol or Acetate

Acetogen strain Clostridium sp. MT1121 produced 300?mM acetate (p?<?0.005) and 321?mM ethanol (p?<?0.005) from synthesis gas (syngas) blend 60?% CO and 40?%?H₂. Clostridium sp. MT1121 was metabolically engineered to eliminate production of either acetate or acetaldehyde during syngas fermentation. We used Cre-lox66/lox71-based gene removal system to eliminate either phosphotransacetylase (pta), or acetaldehyde dehydrogenase (aldh). The resulted biocatalyst with eliminated pta increased ethanol yield to 610?mM (p?<?0.005). Inactivation of pta rendered only 502?mM of ethanol (p?<?0.005). The acetogen biocatalyst with eliminated aldh produced 450?mM acetate (p?<?0.005). The role of cell energy pool preservation for re-directed carbon flux is discussed. This is the first report on time- and cost-efficient gene elimination in acetogens using lox66/lox71 gene elimination system.     

A DNA-based piezoelectric biosensor: strategies for coupling nucleic acids to piezoelectric devices

A DNA-based piezoelectric biosensor has been here studied in terms of probe immobilisation and DNA sample pre-treatment. The biosensor is specific for the detection of the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA).Methicillin-resistant S. aureus is responsible of several infections in humans, like pneumonia, meningitis and endocarditic. MRSA is also a major cause of hospital-acquired infections worldwide. The antibiotics resistance is conferred by the gene mecA, codifying for an anomalous protein.Two different immobilisation procedures of the probe specific for mecA gene are reported: immobilisation via streptavidin-biotin interaction and direct immobilisation of thiolated probes.After the study with synthetic oligonucleotides, the system has been applied to the analysis of bacterial DNA from MRSA, amplified by polymerase chain reaction. These samples were pre-treated with two different denaturation procedures and the performances of the sensor in the two cases were compared.The two immobilisation methods and denaturation protocols were here used to study the influences of these parameters on the performances of the sensor, applied here to the detection of the mecA gene. Better results in terms of sensitivity and reproducibility were obtained when using the biotinylated probe and the PCR-amplified samples treated by a denaturation procedures involving the use of high temperature and blocking oligonucleotides.     

Population Analysis of Mesophilic Microbial Fuel Cells Fed with Carbon Monoxide

Electricity generation in a microbial fuel cell (MFC) fed with carbon monoxide (CO) has been recently demonstrated; however, the microbial ecology of this system has not yet been described. In this work the diversity of the microbial community present at the anode of CO-fed MFCs was studied by performing denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (HTS) analyses. HTS indicated a significant increase of the archaeal genus Methanobacterium and of the bacterial order Clostridiales, notably including Clostridium species, while in both MFCs DGGE identified members of the bacterial genera Geobacter, Desulfovibrio, and Clostridium, and of the archaeal genera Methanobacterium, Methanofollis, and Methanosaeta. In particular, the presence of Geobacter sulfurreducens was identified. Tolerance of G. sulfurreducens to CO was confirmed by growing G. sulfurreducens with acetate under a 100 % CO atmosphere. This observation, along with the identification of acetogens, supports the hypothesis of the two-step process in which CO is converted to acetate by the carboxidotrophic Bacteria and acetate is then oxidized by CO-tolerant electricigenic Bacteria to produce electricity.     

Qualitative and quantitative analysis of polycyclic polyprenylated acylphloroglucinols from Garcinia species using ultra performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Polycyclic polyprenylated acylphloroglucinols (PPAPs) are a group of natural products isolated from different Garcinia species with a wide range of important biological activities. In this study, an ultra performance liquid chromatography (UPLC) coupled to photodiode-array detection and quadrupole time-of-flight mass spectrometry (Q-TOF) method was developed to characterize 16 PPAPs in 10 Garcinia species. In source dissociation techniques based on cone voltage fragmentation were used to fragment the deprotonated molecules and multiple mass spectrometry (MS/MS) using ramping collision energy were used to further break down the resulting product ions. The resulting characteristic fragment ions were generated by cleavage of C1-C5 bond and C7-C8 bond through concerted pericyclic reaction, which is especially valuable for differentiating three types of PPAPs isomers. As such, two new PPAPs isomers present in minor amount in the extracts of Garcinia oblongifolia were tentatively characterized by comparing their tandem mass spectra to the known ones. In addition, an UPLC-Q-TOF-MS method was validated for the quantitative determination of PPAPs. The method exhibited limits of detection from 2.7 to 21.4 ng mL⁻¹ and intra-day and inter-day variations were less than 3.7% and the recovery was in the range of 89-107% with RSD less than 9.0%. This UPLC-Q-TOF-MS method has successfully been applied to quantify 16 PPAPs in 32 samples of 10 Garcinia species, which were found to be a rich source of PPAPs.     

Direct Conversion of Sugars and Organic Acids to Biobutanol by Non-growing Cells of Clostridium spp. Incubated in a Nitrogen-Free Medium

Several Clostridium spp. were incubated in a nitrogen-free medium (non-growth medium) containing only butyric acid as a sole precursor for performing butanol production by non-growing cells. Non-growing cells of Clostridium spp., especially Clostridium beijerinckii TISTR 1461, could convert butyric acid to butanol via their sole solventogenic activity. This activity was further enhanced in the presence of glucose as a co-substrate. In addition to glucose, other monosaccharides (i.e., galactose and xylose) and disaccharides (i.e., maltose, sucrose, and lactose) could also be used as a co-substrate with butyric acid. Among the organic acids tested (i.e., formic, acetic, propionic, and butyric acids), only butyric and acetic acids were converted to butanol. This study has shown that it is possible to use the non-growing cells of Clostridium spp. for direct conversion of sugars and organic acids to biobutanol. With this strategy, C. beijerinckii TISTR 1461 produced 12 g/L butanol from 15 g/L glucose and 10 g/L butyric acid with a high butanol yield of 0.68 C-mol/C-mol and a high butanol ratio of 88 %.     

Pharmacological Insights into Halophyte Bioactive Extract Action on Anti-Inflammatory,Pain Relief and Antibiotics-Type Mechanisms

The pharmacological activities in bioactive plant extracts play an increasing role in sustainable resources for valorization and biomedical applications. Bioactive phytochemicals, including natural compounds, secondary metabolites and their derivatives, have attracted significant attention for use in both medicinal products and cosmetic products. Our review highlights the pharmacological mode-of-action and current biomedical applications of key bioactive compounds applied as anti-inflammatory, bactericidal with antibiotics effects, and pain relief purposes in controlled clinical studies or preclinical studies. In this systematic review, the availability of bioactive compounds from several salt-tolerant plant species, mainly focusing on the three promising species Aster tripolium, Crithmum maritimum and Salicornia europaea, are summarized and discussed. All three of them have been widely used in natural folk medicines and are now in the focus for future nutraceutical and pharmacological applications.     

Effect of Inoculated Lactic Acid Fermentation on the Fermentable Saccharides and Polyols,Polyphenols and Antioxidant Activity Changes in Wheat Sourdough

Inoculation of sourdough allows the fermentation medium to be dominated by desired microorganisms, which enables determining the kinetics of the conversion of chemical compounds by individual microorganisms. This knowledge may allow the design of functional food products with health features dedicated to consumers with special needs. The aim of the study was to assess the dynamics of transformations of fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FODMAP) compounds from wheat flour as well as their antioxidant activity during inoculated and spontaneous sourdough fermentation. The FODMAP content in grain products was determined by the fructan content with negligible amounts of sugars and polyols. To produce a low-FODMAP cereal product, the fermentation time is essential. The 72 h fermentation time of L. plantarum-inoculated sourdough reduced the FODMAP content by 91%. The sourdough fermentation time of at least 72 h also positively influenced the content of polyphenols and antioxidant activity, regardless of the type of fermentation. The inoculation of both L. plantarum and L. casei contributed to a similar degree to the reduction in FODMAP in sourdough compared to spontaneous fermentation.     

Butanol Tolerance in a Selection of Microorganisms

Butanol tolerance is a critical factor affecting the ability of microorganisms to generate economically viable quantities of butanol. Current Clostridium strains are unable to tolerate greater than 2% 1-butanol thus membrane or gas stripping technologies to actively remove butanol during fermentation are advantageous. To evaluate the potential of alternative hosts for butanol production, we screened 24 different microorganisms for their tolerance to butanol. We found that in general, a barrier to growth exists between 1% and 2% butanol and few microorganisms can tolerate 2% butanol. Strains of Escherichia coli, Zymomonas mobilis, and non-Saccharomyces yeasts were unable to surmount the 2% butanol growth barrier. Several strains of Saccharomyces cerevisiae exhibit limited growth in 2% butanol, while two strains of Lactobacillus were able to tolerate and grow in up to 3% butanol.     

Development and Optimisation of HILIC-LC-MS Method for Determination of Carbohydrates in Fermentation Samples

Saccharides are the most common carbon source for Streptococcus thermophilus, which is a widely used bacterium in the production of fermented dairy products. The performance of the strain is influenced by the consumption of different saccharides during fermentation. Therefore, a precise measurement of the concentrations of saccharides in the fermentation media is essential. An 18-min long method with limits of quantitation in the range of 0.159–0.704 mg/L and with ¹³C labelled internal standards employing hydrophilic interaction chromatography coupled to mass spectrometric detection-(HILIC-LC-MS) allowed for simultaneous quantification of five saccharides: fructose, glucose, galactose, sucrose, and lactose in the fermentation samples. The method included a four-step sample preparation protocol, which could be easily applied to high-throughput analysis. The developed method was validated and applied to the fermentation samples produced by Streptococcus thermophilus.     

Fermentation condition outweighed truffle species in affecting volatile organic compounds analyzed by chromatographic fingerprint system

The influences of fermentation conditions and truffle species (i.e., Tuber melanosporum, Tuber sinense, Tuber indicum, and Tuber aestivum) on the volatile organic compounds (VOCs) originated from truffle fermentation mycelia were studied by using chromatographic fingerprint system for the first time. Gas chromatography combined with statistical methods including similarity analysis and hierarchical cluster analysis was applied to develop chromatographic fingerprint system for truffle VOCs evaluation. Fermentation conditions affected the VOCs from truffle fermentation mycelia much more significantly than truffle species. This indicated that it is possible to adjust the aroma of truffle fermentation mycelia similar with the natural fruiting-body through the control of fermentation process.     

Curing of N,N′-diglycidylimides with polyfunctional compounds

This paper describes the curing reactions of new N,N′-diglycidylimides containing trimellitimide units. Reactions were carried out using several polyfunctional curing agents, viz. aromatic primary diamines and phthalic anhydride. Spectroscopic techniques were used to study the curing process. The course of reaction was followed by i.r. spectroscopy. Some intermediate species were detected by recording ¹³C-NMR spectra until the products became insoluble in the usual deuterated solvents.     

Piper betle (L): Recent Review of Antibacterial and Antifungal Properties,Safety Profiles,and Commercial Applications

Piper betle (L) is a popular medicinal plant in Asia. Plant leaves have been used as a traditional medicine to treat various health conditions. It is highly abundant and inexpensive, therefore promoting further research and industrialization development, including in the food and pharmaceutical industries. Articles published from 2010 to 2020 were reviewed in detail to show recent updates on the antibacterial and antifungal properties of betel leaves. This current review showed that betel leaves extract, essential oil, preparations, and isolates could inhibit microbial growth and kill various Gram-negative and Gram-positive bacteria as well as fungal species, including those that are multidrug-resistant and cause serious infectious diseases. P. betle leaves displayed high efficiency on Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, Gram-positive bacteria such as Staphylococcus aureus, and Candida albicans. The ratio of MBC/MIC indicated bactericidal and bacteriostatic effects of P. betle leaves, while MFC/MIC values showed fungicidal and fungistatic effects. This review also provides a list of phytochemical compounds in betel leaves extracts and essential oils, safety profiles, and value-added products of betel leaves. Some studies also showed that the combination of betel leaves extract and essential oil with antibiotics (streptomycin, chloramphenicol and gentamicin) could provide potentiating antibacterial properties. Moreover, this review delivers a scientific resume for researchers in respected areas and manufacturers who want to develop betel leaves-based products.     

Applications and Restrictions of Integrated Genomic and Metabolomic Screening: An Accelerator for Drug Discovery from Actinomycetes?

Since the golden age of antibiotics in the 1950s and 1960s actinomycetes have been the most prolific source for bioactive natural products. However, the number of discoveries of new bioactive compounds decreases since decades. New procedures (e.g., activating strategies or innovative fermentation techniques) were developed to enhance the productivity of actinomycetes. Nevertheless, compound identification remains challenging among others due to high rediscovery rates. Rapid and cheap genome sequencing as well as the advent of bioinformatical analysis tools for biosynthetic gene cluster identification in combination with mass spectrometry-based molecular networking facilitated the tedious process of dereplication. In recent years several studies have been dedicated to accessing the biosynthetic potential of Actinomyces species, especially streptomycetes, by using integrated genomic and metabolomic screening in order to boost the discovery rate of new antibiotics. This review aims to present the various possible applications of this approach as well as the newly discovered molecules, covering studies between 2014 and 2021. Finally, the effectiveness of this approach with regard to find new bioactive agents from actinomycetes will be evaluated.     

Engineering Escherichia coli for Fermentative Dihydrogen Production: Potential Role of NADH-Ferredoxin Oxidoreductase from the Hydrogenosome of Anaerobic Protozoa

Trichomonas vaginalis generates reduced ferredoxin within a unique subcellular organelle, hydrogenosome that is used as a reductant for H₂ production. Pyruvate ferredoxin oxidoreductase and NADH dehydrogenase (NADH-DH) are the two enzymes catalyzing the production of reduced ferredoxin. The genes encoding the two subunits of NADH-DH were cloned and expressed in Escherichia coli. Kinetic properties of the recombinant heterodimer were similar to that of the native enzyme from the hydrogenosome. The recombinant holoenzyme contained 2.15 non-heme iron and 1.95 acid-labile sulfur atoms per heterodimer. The EPR spectrum of the dithionite-reduced protein revealed a [2Fe–2S] cluster with a rhombic symmetry of g _xyz?=?1.917, 1.951, and 2.009 corresponding to cluster N1a of the respiratory complex I. Based on the Fe content, absorption spectrum, and the EPR spectrum of the purified small subunit, the [2Fe–2S] cluster was located in the small subunit of the holoenzyme. This recombinant NADH-DH oxidized NADH and reduced low redox potential electron carriers, such as viologen dyes as well as Clostridium ferredoxin that can couple to hydrogenase for H₂ production from NADH. These results show that this unique hydrogenosome NADH dehydrogenase with a critical role in H₂ evolution in the hydrogenosome can be produced with near-native properties in E. coli for metabolic engineering of the bacterium towards developing a dark fermentation process for conversion of biomass-derived sugars to H₂ as an energy source.     

Extraction,Isolation and Characterization of Bioactive Compounds from Artemisia and Their Biological Significance: A Review

Diverse medicinal plants such as those from the genus Artemisia have been employed globally for centuries by individuals belonging to different cultures. Universally, Artemisia species have been used to remedy various maladies that range from simple fevers to malaria. A survey conducted by the World Health Organization (WHO) demonstrated that 80% of the global population is highly reliant on herbal medicine for their primary healthcare. WHO recommends artemisinin-based combination therapies (ACT) for the treatment of global diseases such as malaria. Artemisinin is a bioactive compound derived from Artemisia annua leaves. It is a sesquiterpene endoperoxide with potent antimalarial properties. This review strives to instill natural products to chemists and others in diverse fields with a heterogeneous set of knowledge compiled from multifaceted researchers and organizations in literature. In particular, the various Artemisia species and effective extraction, isolation, and characterization methodologies are discussed in detail. An in-depth investigation into the literature reveals that divergent species of Artemisia exhibit a vast array of biological activities such as antimalarial, antitumor, and anti-inflammatory activities. There is substantial potential for bioactive compounds from Artemisia to provide significant relief from differing human ailments, but more meticulous research in this field is needed.     

The Wonderful Activities of the Genus Mentha: Not Only Antioxidant Properties

Medicinal plants and their derived compounds have drawn the attention of researchers due to their considerable impact on human health. Among medicinal plants, mint (Mentha species) exhibits multiple health beneficial properties, such as prevention from cancer development and anti-obesity, antimicrobial, anti-inflammatory, anti-diabetic, and cardioprotective effects, as a result of its antioxidant potential, combined with low toxicity and high efficacy. Mentha species are widely used in savory dishes, food, beverages, and confectionary products. Phytochemicals derived from mint also showed anticancer activity against different types of human cancers such as cervix, lung, breast and many others. Mint essential oils show a great cytotoxicity potential, by modulating MAPK and PI3k/Akt pathways; they also induce apoptosis, suppress invasion and migration potential of cancer cells lines along with cell cycle arrest, upregulation of Bax and p53 genes, modulation of TNF, IL-6, IFN-γ, IL-8, and induction of senescence phenotype. Essential oils from mint have also been found to exert antibacterial activities against Bacillus subtilis, Streptococcus aureus, Pseudomonas aeruginosa, and many others. The current review highlights the antimicrobial role of mint-derived compounds and essential oils with a special emphasis on anticancer activities, clinical data and adverse effects displayed by such versatile plants.     

Use of electrophoretic techniques and MALDI–TOF MS for rapid and reliable characterization of bacteria: analysis of intact cells, cell lysates, and “washed pellets”

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and “washed pellets”) were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of “washed pellets” and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI–TOF MS of “washed pellets” enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI–TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.