Advancing Molecular Weight Determination of Lignin by Multi-Angle Light Scattering.

Due to the complexity and recalcitrance of lignin, its chemical characterization is a key factor preventing the valorization of this abundant material. Multi-angle light scattering (MALS) is becoming a sought-after technique for absolute molecular weight (MW) determination of polymers and proteins. Lignin is a suitable candidate for MW determination via MALS, yet further investigation is required to confirm its absolute MW values and molecular size. Studies aiming to break down lignin into a variety of renewable products will benefit greatly from a simple and reliable determination method like MALS. Recent pioneering studies, discussed in this review, addressed several key challenges in lignin's MW characterization. Nevertheless, some lignin-specific issues still need to be considered for in-depth characterization. This study explores how MALS instrumentation manages the complexities of determining lignin's MW, e.g., with simultaneous fractionation and fluorescence interference mitigation. Additionally, we rationalize the importance of a more detailed light scattering analysis for lignin characterization, including aspects like the second virial coefficient and radius of gyration.

Unfolding of Lignin Structure Using Size-Exclusion Fractionation.

The heterogeneous and recalcitrant structure of lignin hinders its practical application. Here, we describe how new approaches to lignin characterization can reveal structural details that could ultimately lead to its more efficient utilization. A suite of methods, which enabled mass balance closure, the evaluation of structural features, and an accurate molecular weight (MW) determination, were employed and revealed unexpected structural features of the five alkali lignin fractions obtained with preparative size-exclusion chromatography (SEC). A thermal carbon analysis (TCA) provided quantitative temperature profiles based on sequential carbon evolution, including the final oxidation of char. The TCA results, supported with thermal desorption/pyrolysis gas chromatography-mass spectrometry (TD-Py-GC-MS) and 31P NMR spectroscopy, revealed the unfolding of the lignin structure as a result of the SEC fractionation, due to the disruption of the interactions between the high- and low-MW components. The "unraveled" lignin revealed poorly accessible hydroxyl groups and showed an altered thermal behavior. The fractionated lignin produced significantly less char upon pyrolysis, 2 vs. 47%. It also featured a higher occurrence of low-MW thermal evolution products, particularly guaiacol carbonyls, and more than double the number of OH groups accessible for phosphitylation. These observations indicate pronounced alterations in the lignin intermolecular association following size-exclusion fractionation, which may be used for more efficient lignin processing in biorefineries.

A Comprehensive Study of Techniques to Optimize the Extraction of Lipids from the Autotrophic Strain of the Microalgae Chlorella vulgaris.

Microalgae represent a promising source of triglycerides and free fatty acids, synthesized in the form of lipids, for use in renewable fuels and chemicals. One challenge is the ability to efficiently recover the lipids from within the microalgae cell. Although various techniques have been studied individually, a comprehensive study of extraction techniques using consistent experimental and analytical methodology is missing. This study aims to provide this unifying comparison using the common microalgae strain Chlorella vulgaris. The factors that were surveyed and then optimized to achieve maximum extraction efficiency included the solvent type; mechanical pre-treatment using a ball mill at a variety of grinding speeds; microalgae-to-solvent ratio; extraction facilitated by microwave; extraction facilitated by sonication; extraction facilitated using increased temperature; and extraction facilitated by in situ transesterification to convert the lipids into esters prior to extraction. The optimum conditions determined during these studies were utilizing methanol as the solvent, with ball mill pretreatment at a grinding speed of 500 rpm, and a 1:9 microalgae to solvent ratio. When used in combination with microwave-assisted extraction at a temperature of 140 °C, approximately 24 wt% of the initial lipids were recovered. Recoveries of over 70 wt% were obtained without a microwave at extraction temperatures of over 200 °C.

A visceral mycosis in farmed rainbow trout (Oncorhynchus mykiss) caused by Neopyrenochaeta submersa.

A mycotic infection manifesting as abdominal distension with free serous fluid accumulation in the coelomic cavity is documented in farmed rainbow trout. Histological examination using PAS and silver staining revealed the presence of numerous fungal hyphae in the spleen and gastrointestinal wall. The isolated fungus was sterile and identified by using phylogenetic analysis based on four loci as Neopyrenochaeta submersa. This is the first time this fungus has been reported as pathogen.

Mechanistic Investigations of Thermal Decomposition of Perfluoroalkyl Ether Carboxylic Acids and Short-Chain Perfluoroalkyl Carboxylic Acids.

In this study, we investigated the thermal decomposition mechanisms of perfluoroalkyl ether carboxylic acids (PFECAs) and short-chain perfluoroalkyl carboxylic acids (PFCAs) that have been manufactured as replacements for phased-out per- and polyfluoroalkyl substances (PFAS). C-C, C-F, C-O, O-H, and C═C bond dissociation energies were calculated at the M06-2X/Def2-TZVP level of theory. The α-C and carboxyl-C bond dissociation energy of PFECAs declines with increasing chain length and the attachment of an electron-withdrawing trifluoromethyl (-CF3) group to the α-C. Experimental and computational results show that the thermal transformation of hexafluoropropylene oxide dimer acid to trifluoroacetic acid (TFA) occurs due to the preferential cleavage of the C-O ether bond close to the carboxyl group. This pathway produces precursors of perfluoropropionic acid (PFPeA) and TFA and is supplemented by a minor pathway (CF3CF2CF2OCFCF3COOH → CF3CF2CF2· + ·OCFCF3COOH) through which perfluorobutanoic acid (PFBA) is formed. The weakest C-C bond in PFPeA and PFBA is the one connecting the α-C and the ß-C. The results support (1) the C-C scission in the perfluorinated backbone as an effective PFCA thermal decomposition mechanism and (2) the thermal recombination of radicals through which intermediates are formed. Additionally, we detected a few novel thermal decomposition products of studied PFAS.

Effects of organic chemicals from diesel exhaust particles on adipocytes differentiated from human mesenchymal stem cells.

Exposure to fine particulate matter (PM2.5 ) from incomplete fossil fuel combustion (coal, oil, gas and diesel) has been linked to increased morbidity and mortality due to metabolic diseases. PM2.5 exaggerate adipose inflammation and insulin resistance in mice with diet-induced obesity. Here, we elucidate the hypothesis that such systemic effects may be triggered by adhered particle components affecting adipose tissue directly. Studying adipocytes differentiated from primary human mesenchymal stem cells, we found that lipophilic organic chemicals (OC) from diesel exhaust particles induced inflammation-associated genes and increased secretion of the chemokine CXLC8/interleukin-8 as well as matrix metalloprotease 1. The oxidative stress response gene haem oxygenase-1 and tumour necrosis factor alpha were seemingly not affected, while aryl hydrocarbon receptor-regulated genes, cytochrome P450 1A1 (CYP1A1) and CYP1B1 and plasminogen activator inhibitor-2, were clearly up-regulated. Finally, expression of ß-adrenergic receptor, known to regulate adipocyte homoeostasis, was down-regulated by exposure to these lipophilic OC. Our results indicate that low concentrations of OC from combustion particles have the potential to modify expression of genes in adipocytes that may be linked to metabolic disease. Further studies on mechanisms linking PM exposure and metabolic diseases are warranted.

Road tunnel-derived coarse, fine and ultrafine particulate matter: physical and chemical characterization and pro-inflammatory responses in human bronchial epithelial cells.

BACKGROUND: Traffic particulate matter (PM) comprises a mixture of particles from fuel combustion and wear of road pavement, tires and brakes. In countries with low winter temperatures the relative contribution of mineral-rich PM from road abrasion may be especially high due to use of studded tires during winter season. The aim of the present study was to sample and characterize size-fractioned PM from two road tunnels paved with different stone materials in the asphalt, and to compare the pro-inflammatory potential of these fractions in human bronchial epithelial cells (HBEC3-KT) in relation to physicochemical characteristics. METHODS: The road tunnel PM was collected with a vacuum pump and a high-volume cascade impactor sampler. PM was sampled during winter, both during humid and dry road surface conditions, and before and after cleaning the tunnels. Samples were analysed for hydrodynamic size distribution, content of elemental carbon (EC), organic carbon (OC) and endotoxin, and the capacity for acellular generation of reactive oxygen species. Cytotoxicity and pro-inflammatory responses were assessed in HBEC3-KT cells after exposure to coarse (2.5-10 µm), fine (0.18-2.5 µm) and ultrafine PM (≤ 0.18 µm), as well as particles from the respective stone materials used in the pavement. RESULTS: The pro-inflammatory potency of the PM samples varied between road tunnels and size fractions, but showed more marked responses than for the stone materials used in asphalt of the respective tunnels. In particular, fine samples showed significant increases as low as 25 µg/mL (2.6 µg/cm2) and were more potent than coarse samples, while ultrafine samples showed more variable responses between tunnels, sampling conditions and endpoints. The most marked responses were observed for fine PM sampled during humid road surface conditions. Linear correlation analysis showed that particle-induced cytokine responses were correlated to OC levels, while no correlations were observed for other PM characteristics. CONCLUSIONS: The pro-inflammatory potential of fine road tunnel PM sampled during winter season was high compared to coarse PM. The differences between the PM-induced cytokine responses were not related to stone materials in the asphalt. However, the ratio of OC to total PM mass was associated with the pro-inflammatory potential.

The first quantitative investigation of compounds generated from PFAS, PFAS-containing aqueous film-forming foams and commercial fluorosurfactants in pyrolytic processes.

Pyrolysis as a thermochemical technology is commonly used in waste management and remediation of organic-contaminated soil. This study, for the first time, investigated fluorinated and non-fluorinated compounds emitted from per- and polyfluoroalkyl substances (PFAS) and relevant products upon pyrolysis (200-890 °C) and their formation mechanisms. Approximately 30 non-fluorinated compounds were detected from PFAS-containing aqueous film-forming foams (AFFFs) and commercial surfactant concentrates (SCs) after heating, including glycols and glycol ethers that were predominant at 200 °C. Oxygen (e.g., 1,4-dioxane) and nitrogen heterocycles and benzene were unexpectedly observed at higher temperatures (300-890 °C), which were likely formed as a consequence of the thermal dehydration, dehydrogenation, and intermolecular cyclization of glycols and glycol ethers. Fluorinated volatiles in six major classes were detected at low and moderate temperatures (200-500 °C), including perfluoroalkenes, perfluoroalkyl aldehydes, fluorotelomer alcohols, and polyfluorinated alkanes/alkenes. Several features of the pyrolyses of PFAS suggest that the underlying decomposition mechanism is radical-mediated. Perfluoroheptene thermally decomposed at 200 °C to shorter-chain homologues following a radical chain-scission mechanism. Most of these volatiles observed at low/moderate temperatures were not detected at 890 °C. Ultra-short-chain fluorinated greenhouse gases (e.g., perfluoromethane) were not found.

Hybrid Synthetic and Computational Study of an Optimized, Solvent-Free Approach to Curcuminoids.

A green and optimized protocol has been developed for the preparation of symmetric 1,7-bis(aryl)-1,6-heptadiene-3,5-diones and asymmetric 2-aryl-6-arylidenecyclohexanones with modified substrate scope and good functional group tolerance. Syntheses proceed smoothly under solvent-free conditions, providing moderate to excellent product yields with a minimal workup procedure. Control experiments, spectroscopic, and computational studies support a mechanism involving the boron-assisted in situ generation of imine intermediates. Crystal structures of three curcuminoids and isolated mechanistic intermediates are reported. The data provide insight for the further development of solvent-free protocols toward diverse curcumin derivatives in the fields of pharmaceutical and synthetic chemistries.

The Transgenerational Transmission of the Paternal Type 2 Diabetes-Induced Subfertility Phenotype.

Diabetes is a chronic metabolic disorder characterized by hyperglycemia and associated with many health complications due to the long-term damage and dysfunction of various organs. A consequential complication of diabetes in men is reproductive dysfunction, reduced fertility, and poor reproductive outcomes. However, the molecular mechanisms responsible for diabetic environment-induced sperm damage and overall decreased reproductive outcomes are not fully established. We evaluated the effects of type 2 diabetes exposure on the reproductive system and the reproductive outcomes of males and their male offspring, using a mouse model. We demonstrate that paternal exposure to type 2 diabetes mediates intergenerational and transgenerational effects on the reproductive health of the offspring, especially on sperm quality, and on metabolic characteristics. Given the transgenerational impairment of reproductive and metabolic parameters through two generations, these changes likely take the form of inherited epigenetic marks through the germline. Our results emphasize the importance of improving metabolic health not only in women of reproductive age, but also in potential fathers, in order to reduce the negative impacts of diabetes on subsequent generations.

Quantitative insights on de/repolymerization and deoxygenation of lignin in subcritical water.

The objective of the study was to investigate alkali lignin polymerization/depolymerization pathways in subcritical water (SW) without additives. Following a SW treatment at 200, 250, 275 and 300 °C, the products were subjected to a comprehensive suite of analyses addressing the product speciation and molecular weight (MW) distribution. The MW reduction (1.4 times) in the solid products following the SW treatment indicated a surprisingly reduced impact of cross-linking/repolymerization at 300 °C and lower temperatures. This was further confirmed by thermal carbon analysis (TCA) showing a reduction in pyrolytic charring after the SW treatment. The TD-Py gas chromatography analysis of the SW treated lignin indicated that the solid residue is less oxygenated than the initial lignin (23 vs. 29% as confirmed by elemental analysis). Thus, deoxygenation rather than re-polymerization appears to be the main process route in the absence of catalysts within the temperature range considered.

Polyphasic Approach Utilized for the Identification of Two New Toxigenic Members of Penicillium Section Exilicaulis, P. krskae and P. silybi spp. nov.

Two new species, Penicillium krskae (isolated from the air as a lab contaminant in Tulln (Austria, EU)) and Penicillium silybi (isolated as an endophyte from asymptomatic milk thistle (Silybum marianum) stems from Josephine County (Oregon, USA)) are described. The new taxa are well supported by phenotypic (especially conidial ornamentation under SEM, production of red exudate and red pigments), physiological (growth at 37 °C, response to cycloheximide and CREA), chemotaxonomic (production of specific extrolites), and multilocus phylogenetic analysis using RNA-polymerase II second largest subunit (RPB2), partial tubulin (benA), and calmodulin (CaM). Both new taxa are resolved within the section Exilicaulis in series Restricta and show phylogenetic affiliation to P. restrictum sensu stricto. They produce a large spectrum of toxic anthraquinoid pigments, namely, monomeric anthraquinones related to emodic and chloremodic acids and other interesting bioactive extrolites (i.e., endocrocin, paxilline, pestalotin, and 7-hydroxypestalotin). Of note, two bianthraquinones (i.e., skyrin and oxyskyrin) were detected in a culture extract of P. silybi. Two new chloroemodic acid derivatives (2-chloro-isorhodoptilometrin and 2-chloro-desmethyldermoquinone) isolated from the exudate of P. krskae ex-type culture were analyzed by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS).

Molecular systematics of Keratinophyton: the inclusion of species formerly referred to Chrysosporium and description of four new species.

Four new Keratinophyton species (Ascomycota, Pezizomycotina, Onygenales), K. gollerae, K. lemmensii, K. straussii, and K. wagneri, isolated from soil samples originating from Europe (Austria, Italy, and Slovakia) are described and illustrated. The new taxa are well supported by phylogenetic analysis of the internal transcribed spacer region (ITS) region, the combined data analysis of ITS and the nuclear large subunit (LSU) rDNA, and their phenotype. Based on ITS phylogeny, within the Keratinophyton clade, K. lemmensii is clustered with K. durum, K. hubeiense, K. submersum, and K. siglerae, while K. gollerae, K. straussii and K. wagneri are resolved in a separate terminal cluster. All four new species can be well distinguished from other species in the genus based on phenotype characteristics alone. Ten new combinations are proposed for Chrysosporium species which are resolved in the monophyletic Keratinophyton clade. A new key to the recognized species is provided herein.

Effect of granular activated carbon and other porous materials on thermal decomposition of per- and polyfluoroalkyl substances: Mechanisms and implications for water purification.

Thermal treatment is routinely used to reactivate the spent granular activated carbon (GAC) from water purification facilities. It is also an integral part of sewage sludge treatment and municipal solid waste management. This study presents a detailed investigation of the fate of per- and polyfluoroalkyl substances (PFAS) and one PFAS alternative (GenX) in thermal processes, focusing on the effect of GAC. We demonstrate that the thermolysis of perfluoroalkyl carboxylic acids (PFCAs), including perfluorooctanoic acid (PFOA), and GenX can occur at temperatures of 150‒200 °C. Three temperature zones were discovered for PFOA, including a stable and nonvolatile zone (≤90 °C), a phase-transfer and thermal decomposition zone (90‒400 °C), and a fast decomposition zone (≥400 °C). The thermal decomposition began with the homolysis of a C‒C bond next to the carboxyl group of PFCAs, which formed unstable perfluoroalkyl radicals. Dual decomposition pathways seem to exist. The addition of a highly porous adsorbent, such as GAC or a copolymer resin, compressed the intermediate sublimation zone of PFCAs, changed their thermal decomposition pathways, and increased the decomposition rate constant by up to 150-fold at 250 °C. The results indicate that the observed thermal decomposition acceleration was linked to the adsorption of gas-phase PFCA molecules on GAC. The presence of non-activated charcoals/biochars with a low affinity for PFOA did not accelerate its thermal decomposition, suggesting that the π electron-rich, polyaromatic surface of charcoal/GAC played an insignificant role compared to the adsorbent's porosity. Overall, the results indicate that (1) substantial decomposition of PFCAs and GenX during conventional thermal GAC/sludge/waste treatment is very likely, and (2) the presence or addition of GAC or other highly porous materials can accelerate thermal PFAS decomposition and alter decomposition pathways.

Comparative eco-physiology revealed extensive enzymatic curtailment, lipases production and strong conidial resilience of the bat pathogenic fungus Pseudogymnoascus destructans.

The genus Pseudogymnoascus encompasses soil psychrophilic fungi living also in caves. Some are opportunistic pathogens; nevertheless, they do not cause outbreaks. Pseudogymnoascus destructans is the causative agent of the white-nose syndrome, which is decimating cave-hibernating bats. We used comparative eco-physiology to contrast the enzymatic potential and conidial resilience of P. destructans with that of phylogenetically diverse cave fungi, including Pseudogymnoascus spp., dermatophytes and outdoor saprotrophs. Enzymatic potential was assessed by Biolog MicroArray and by growth on labelled substrates and conidial viability was detected by flow cytometry. Pseudogymnoascus destructans was specific by extensive losses of metabolic variability and by ability of lipid degradation. We suppose that lipases are important enzymes allowing fungal hyphae to digest and invade the skin. Pseudogymnoascus destructans prefers nitrogenous substrates occurring in bat skin and lipids. Additionally, P. destructans alkalizes growth medium, which points to another possible virulence mechanism. Temperature above 30 °C substantially decreases conidial viability of cave fungi including P. destructans. Nevertheless, survival of P. destructans conidia prolongs by the temperature regime simulating beginning of the flight season, what suggests that conidia could persist on the body surface of bats and contribute to disease spreading during bats active season.

Pathways toward PAH Formation during Fatty Acid and Triglyceride Pyrolysis.

Molecular beam mass spectrometry was used to follow model triglyceride pyrolysis with temperature. A selectively formed set of PAHs (276, 352, 444 amu) arose with increasing temperature. They were attributed to association of up to five C7-C8 sized fragments (observed in abundance by pyrolysis with gas chromatography), presumably due to their propensity to form stable benzyl radicals. Results were surprisingly similar regardless of triglyceride fatty acids (FAs), containing 0-2 C=C double bonds (14 to 18 carbon atoms). However, the absence of C=C double bonds shifted the process to higher temperatures. Shorter FA chains, particularly 14:0, enhanced generation of shorter size fragments, facilitating the alternate formation of nonselective PAH homology series. An increase in the length of the glass wool filled sample stage enhanced the formation of two more PAHs, 316 and 388 amu. They appear to involve the formation of indenyl in addition to benzyl radicals as key intermediates.

A common partitivirus infection in United States and Czech Republic isolates of bat white-nose syndrome fungal pathogen Pseudogymnoascus destructans.

The psychrophilic (cold-loving) fungus Pseudogymnoascus destructans was discovered more than a decade ago to be the pathogen responsible for white-nose syndrome, an emerging disease of North American bats causing unprecedented population declines. The same species of fungus is found in Europe but without associated mortality in bats. We found P. destructans was infected with a mycovirus [named Pseudogymnoascus destructans partitivirus 1 (PdPV-1)]. The virus is bipartite, containing two double-stranded RNA (dsRNA) segments designated as dsRNA1 and dsRNA2. The cDNA sequences revealed that dsRNA1 dsRNA is 1,683 bp in length with an open reading frame (ORF) that encodes 539 amino acids (molecular mass of 62.7 kDa); dsRNA2 dsRNA is 1,524 bp in length with an ORF that encodes 434 amino acids (molecular mass of 46.9 kDa). The dsRNA1 ORF contains motifs representative of RNA-dependent RNA polymerase (RdRp), whereas the dsRNA2 ORF sequence showed homology with the putative capsid proteins (CPs) of mycoviruses. Phylogenetic analyses with PdPV-1 RdRp and CP sequences indicated that both segments constitute the genome of a novel virus in the family Partitiviridae. The purified virions were isometric with an estimated diameter of 33 nm. Reverse transcription PCR (RT-PCR) and sequencing revealed that all US isolates and a subset of Czech Republic isolates of P. destructans were infected with PdPV-1. However, PdPV-1 appears to be not widely dispersed in the fungal genus Pseudogymnoascus, as non-pathogenic fungi P. appendiculatus (1 isolate) and P. roseus (6 isolates) tested negative. P. destructans PdPV-1 could be a valuable tool to investigate fungal biogeography and the host-pathogen interactions in bat WNS.

Gestational and pubertal exposure to low dose of di-(2-ethylhexyl) phthalate impairs sperm quality in adult mice.

Di-(2-ethylhexyl)-phthalate (DEHP) is a compound widely used as a plasticizer, which can leach from plastics into the environment and thus influence human health. The aim of this study was to analyze whether exposure to an environmentally relevant dose of DEHP during mice fetal development or puberty can cause long-lasting changes detectable month/s after the last exposure. We used a DEHP concentration relevant to a daily human intake of 2.4-3 µg/kg of body weight/day. CD1 outbred mice were treated either in utero or postnatally during puberty and analyzed in adulthood. Analyzing fertility parameters using morphometric, histologic, genomic and proteomic methods we showed that DEHP exposure leads to decreased sperm concentration and quality, in both experimental groups. Moreover, the changes in anogenital distance, seminal vesicle weight, and testicular gene expression suggest a disturbance of androgen signaling in exposed animals. In conclusion, we hereby present, that the prenatal and pubertal exposure to a low dose of DEHP negatively influenced reproductive endpoints in male mice, and some of the effects were persistent until adulthood.

Swim bladder mycosis in farmed rainbow trout Oncorhynchus mykiss caused by Phoma herbarum and experimental verification of pathogenicity.

In this study, spontaneous swim bladder mycosis was documented in a farmed fingerling rainbow trout from a raceway culture system. At necropsy, the gross lesions included a thickened swim bladder wall, and the posterior portion of the swim bladder was enlarged due to massive hyperplasia of muscle. A microscopic wet mount examination of the swim bladder contents revealed abundant septate hyphae, and histopathological examination showed periodic acid-Schiff-positive mycelia in the lumen and wall of the swim bladder. Histopathological examination of the thickened posterior swim bladder revealed muscle hyperplasia with expansion by inflammatory cells. The causative agent was identified as Phoma herbarum through morphological analysis and DNA sequencing. The disease was reproduced in rainbow trout fingerlings using intraperitoneal injection of a spore suspension. Necropsy in dead and moribund fish revealed extensive congestion and haemorrhages in the serosa of visceral organs and in liver and abdominal serosanguinous fluid. Histopathological examination showed severe hepatic congestion, sinusoidal dilatation, Kupffer cell reactivity, leukostasis and degenerative changes. Fungi were disseminated to the liver, pyloric caeca, kidney, spleen and heart. Although infections caused by Phoma spp. have been repeatedly reported in fish, species identification has been hampered by extensive taxonomic changes. The results of this study confirmed the pathogenicity of P. herbarum in salmonids by using a reliably identified strain during experimental fish infection and provides new knowledge regarding the course of infection.

Atmospheric pressure ionization mass spectrometry as a tool for structural characterization of lignin.

RATIONALE: Lignin occurs in a broad range of forms, e.g., native as the main support for plant walls, and processed, for which its structure depends on the nature of the industrial isolation method, such as in paper production or in biorefineries. Due to the variety of lignin sources, there is no unified agreement on the structure of lignin or even its molecular weight (MW). METHODS: The focus of this review is on the application of atmospheric pressure ionization methods to lignin analysis by mass spectrometry (MS), namely electrospray ionization (ESI) or direct analysis in real-time (DART). Specific parameters affecting ionization including electrolytes and solvents are discussed. RESULTS: The main challenge for MW determination of lignin is its heteropolymer character as well as the mass range limitations of MS instrumentation. To date, only a few studies have successfully used the mass range above m/z 1500. We present the advantage of ESI in generating multiply charged ions, allowing for a further increase in the mass range of deconvoluted mass spectra. While some methods such as DART do not address the mass range problem, they may serve as excellent imaging tools suitable for structural characterization of lignin. CONCLUSIONS: A literature review presents the recent accomplishments in lignin MS analysis by atmospheric pressure ionization techniques. Although significant breakthroughs have been made, it is essential to further improve the operating conditions and validate the methods for a broader range of feedstocks with the results being confirmed using other methods.