Long-term and combined heavy-metal contamination forms a unique microbiome and resistome: A case study in a Yellow River tributary sediments.

Microorganisms have developed mechanisms to adapt to environmental stress, but how microbial communities adapt to long-term and combined heavy-metal contamination under natural environmental conditions remains unclear. Specifically, this study analyzed the characteristics of heavy metal composition, microbial community, and heavy metal resistance genes (MRGs) in sediments along Mang River, a tributary of the Yellow River, which has been heavily polluted by industrial production for more than 40 years. The results showed that the concentrations of Cr, Zn, Pb, Cu and As in most sediments were higher than the ambient background values. Bringing the heavy metals speciation and concentration into the risk evaluation method, two-thirds of the sediment samples were at or above the moderate risk level, and the ecological risk of combined heavy metals in the sediments decreased along the river stream. The high ecological risk of heavy metals affected the microbial community structure, metabolic pathways and MRG distribution. The formation of a HM-resistant microbiome possibly occurred through the spread of insertion sequences (ISs) carrying multiple MRGs, the types of ISs carrying MRGs outnumber those of plasmids, and the quantity of MRGs on ISs is also higher than that on plasmids. These findings could improve our understanding of the adaptation mechanism of microbial communities to long-term combined heavy metal contamination.

Rotenone encapsulated in pH-responsive alginate-based microspheres reduces toxicity to zebrafish.

Rotenone is a botanical pesticide and has long been used for control of insect pests and also as a natural piscicide for management of fish populations in many countries. Field application for pest control, however, often encounters the movement of rotenone into surface water due to spray drift or surface runoff after rainfall, which could potentially result in water pollution and unexpected death of fishes. To minimize its effect on freshwater and the problem of fish dying, one solution was to encapsulate rotenone in specific microspheres, limiting its release and reducing its toxicity since rotenone can be quickly degraded under sunlight. In this study, pH-responsive alginate-based microspheres were synthesized to encapsulating rotenone, which were designated as rotenone beads. The rotenone beads, along with alginate beads (devoid of rotenone) were characterized and evaluated for their responses to pH and effects on zebrafish. Results showed that the microspheres had high loading efficiency (4.41%, w/w) for rotenone, and rotenone beads well responded to solution pH levels. The cumulative release rates of rotenone from the beads were 27.91%, 42.72%, and 90.24% at pH 5.5, 7.0, and 9.0, respectively. Under acidic conditions, the rotenone release rate was lower due to hydrogen bonding. On the contrary, rotenone became more quickly released at the high pH due to intermolecular repulsion. The toxicity of rotenone beads to zebrafish and fish embryos at a pH of 5.5 was reduced by 2- and 4-fold than chemical rotenone. Since pH levels in most freshwater lakes, ponds, and streams vary from 6 to 8, rotenone release from the beads in such freshwater could be limited. Thus, the synthesized rotenone beads could be relatively safely used for pest control with limited effects on freshwater fishers.

The combination of thymol and cinnamaldehyde reduces the survival and virulence of Listeria monocytogenes on autoclaved chicken breast.

AIMS: To reveal the antibacterial mechanism of the combination of thymol and cinnamaldehyde to Listeria monocytogenes ATCC 19115 on autoclaved chicken breast. METHODS AND RESULTS: In this study, L. monocytogenes ATCC 19115 on autoclaved chicken breast was exposed to the stress of 125 µg/ml thymol and 125 µg/ml cinnamaldehyde, and transcriptome analysis was used to reveal the crucial antibacterial mechanism. According to the results, 1303 significantly differentially expressed genes (DEGs) were identified. Treated by thymol and cinnamaldehyde in combination, pyrimidine and branched-chain amino acid biosynthesis of L. monocytogenes were thwarted which impairs its nucleic acid biosynthesis and intracellular metabolism. The up-regulated DEGs involved in membrane composition and function contributed to membrane repair. Besides, pyruvate catabolism and TCA cycle were restrained which brought about the disturbance of amino acid metabolism. ABC transporters were also perturbed, for instance, the uptake of cysteine, D-methionine, and betaine was activated, while the uptake of vitamin, iron, and carnitine was repressed. Thus, L. monocytogenes tended to activate PTS, glycolysis, glycerol catabolism, and pentose phosphate pathways to obtain energy to adapt to the hostile condition. Noticeably, DEGs involved in virulence factors were totally down-regulated, including genes devoted to encoding flagella, chemotaxis, biofilm formation, internalin as well as virulence gene clusters. CONCLUSIONS: The combination of thymol and cinnamaldehyde is effective to reduce the survival and potential virulence of L. monocytogenes on autoclaved chicken breast. SIGNIFICANCE AND IMPACT OF STUDY: This work contributes to providing theoretical information for the application and optimization of thymol and cinnamaldehyde in ready-to-eat meat products to inhibit L. monocytogenes.

Industrial aerobic composting and the addition of microbial agents largely reduce the risks of heavy metal and ARG transfer through livestock manure.

Composting is an effective and necessary modality in the recycling of agricultural wastes such as livestock manure, furfural, and straw. However, the risks of heavy metals (HMs) and antibiotic resistance genes (ARGs) during industrial-scale composting process have not been adequately assessed, especially with the addition of bacterial agents. In this study, changes in HMs toxicity, ARGs propagation and microbial community structure during industrial-scale aerobic composting of livestock manure were firstly investigated with various substrates addition. Moreover, the effect of the addition of bacterial agents (Bacillus strains) was evaluated. The results showed that industrial aerobic composting process could immobilize various HMs with different extents and significantly reduce the levels of ARGs such as intl1 and oqxB genes. The addition of Bacillus strains could further reduce the levels of most detected ARGs and the bioavailability of Cu and Cr, and the relative abundance of ereA and tetA was undetectable in some materials. After composting, the main bacterial community structures were similar among different substrates irrespective of bacterial agents and indicated that the composting process was the main driver for their change. This study provides a scientific reference for the safe reuse of livestock manure.

Preparation of sodium alginate-poly (vinyl alcohol) blend beads for base-triggered release of dinotefuran in Spodoptera litera midgut.

This study investigated the ability of dual crosslinked interpenetrating polymer network (IPN) blend beads (DIN:SA/PVA-beads), composed of sodium alginate (SA) and poly (vinyl alcohol) (PVA), as a base-triggered carrier for the controlled release of dinotefuran (DIN) in Spodoptera litera midgut. The blend beads were characterized for morphology, encapsulation efficiency, swelling degree, and in vitro release of the blend beads were characterized. The results revealed that the double-crosslinked gel beads had a tightly interpenetrating network structure and exhibited a satisfactory embedding effect for DIN. The maximum of the DIN loading capacity was approximately 1.01%, with a high encapsulation efficiency of 83.19%. The triggered release of DIN from the blend beads was studied in deionized water (pH 3.0-11.0) via high-performance liquid chromatography (HPLC); it was found that the release rate was higher in alkaline pH conditions than in acidic and neutral conditions. An in vivo dynamics and degradation study also demonstrated that the excellent release characteristics of DIN:SA/PVA-beads in the midgut of S. litera. This study provides a promising controlled-release form of dinotefuran that is more effective and can be used for the targeted control of pests with alkaline midgut.

Improving Anemia in Inflammatory Bowel Disease: Impact of the Anemia Care Pathway.

BACKGROUND: Anemia is a common complication of inflammatory bowel disease (IBD). Despite existing guidelines for anemia in IBD, it is frequently under-treated and the prevalence of anemia has remained high. To address this gap, the Crohn's and Colitis Foundation developed the Anemia Care Pathway (ACP). AIMS: To implement the ACP in a managed care setting and identify where it improves practice habits and where barriers remain. METHODS: The ACP was implemented from July 2016 through June 2017 and retrospectively studied. Run charts were used to identify shifts in iron deficiency screening and treatment as well as anemia prevalence. Results were compared to those of other providers in the same center not using the ACP. RESULTS: 640 IBD encounters were studied. In the ACP clinic (n = 213), anemics received iron therapy in only 30% of encounters at baseline but improved to 80%. Concurrently, anemia prevalence decreased from 48 to 25%. Screening for iron deficiency, however, did not improve. No shifts were seen in the non-ACP clinics (n = 427) across the same period despite awareness of the ACP and other guidelines. CONCLUSIONS: Across 1 year, we observed gaps in the screening and treatment of anemia in IBD. Although screening rates did not improve, the ACP appeared to reduce missed opportunities for iron therapy by about half. Most importantly, this was associated with an overall decrease in anemia prevalence. Future refinements to the ACP should be focused on enhanced screening and follow-up.

Evolution of Functional Bacteria in a Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Bioreactor.

In this study a laboratory-scale sequencing batch bioreactor was constructed, using phenanthrene/pyrene as the sole carbon-source, to demonstrate the removal efficiencies of polycyclic aromatic hydrocarbons (PAHs) and the evolution process of a functional bacterial community. The removal rates were: COD, 51.4 to 76.3%; phenanthrene, 63.5 to 92.4%; and pyrene, 65.9 to 90.1%. Quantification analysis indicated that in sludge samples from the phenanthrene/pyrene degradation stage, the contents of 16S rRNA and ring-hydroxylating dioxygenase genes of Gram-positive and Gram-negative bacteria were 10-and 72-, 102- and 1152-, 31- and 905-fold higher, respectively, than those in seed sludge samples. Clone sequencing and evolution analysis showed that genera Comamonas, Pseudomonas, and Mycobacterium were distributed across all samples and were significant PAH-degrading populations, but some genera, such as Polaromonas and Nocardioides, were substantially enriched only when phenanthrene/pyrene was fed. Correspondingly, the functional genes nidA3, phnAc, and ndoC2 were distributed across all samples, whereas nagAc, pdoA2, and pdoA were enriched only when phenanthrene/pyrene was fed.

NCAM affects directional lamellipodia formation of BMSCs via ß1 integrin signal-mediated cofilin activity.

The neural cell adhesion molecule (NCAM), a key member of the immunoglobulin-like CAM family, was reported to regulate the migration of bone marrow-derived mesenchymal stem cells (BMSCs). However, the detailed cellular behaviors including lamellipodia formation in the initial step of directional migration remain largely unknown. In the present study, we reported that NCAM affects the lamellipodia formation of BMSCs. Using BMSCs from Ncam knockout mice we found that Ncam deficiency significantly impaired the migration and the directional lamellipodia formation of BMSCs. Further studies revealed that Ncam knockout decreased the activity of cofilin, an actin-cleaving protein, which was involved in directional protrusions. To explore the molecular mechanisms involved, we examined protein tyrosine phosphorylation levels in Ncam knockout BMSCs by phosphotyrosine peptide array analyses, and found that the tyrosine phosphorylation level of ß1 integrin, a protein upstream of cofilin, was greatly upregulated in Ncam-deficient BMSCs. Notably, by blocking the function of ß1 integrin with RGD peptide or ROCK inhibitor, the cofilin activity and directional lamellipodia formation of Ncam knockout BMSCs could be rescued. Finally, we found that the effect of NCAM on tyrosine phosphorylation of ß1 integrin was independent of the fibroblast growth factor receptor. These results indicated that NCAM regulates directional lamellipodia formation of BMSCs through ß1 integrin signal-mediated cofilin activity.

Practical survey on antibiotic-resistant bacterial communities in livestock manure and manure-amended soil.

Through livestock manure fertilization, antibiotics, antibiotic-resistant bacteria and genes are transferred to agricultural soils, resulting in a high prevalence of antibiotic-resistant bacteria in the soil. It is not clear, however, whether a correlation exists between resistant bacterial populations in manure and manure-amended soil. In this work, we demonstrate that the prevalence of cephalexin-, amoxicillin-, kanamycin- and gentamicin-resistant bacteria as well as bacteria simultaneously resistant to all four antibiotics was much higher in manure-amended soils than in manure-free soil. 454-pyrosequencing indicated that the ARB and multiple antibiotic-resistant bacteria (MARB) in swine or chicken manure and manure-amended soil were mainly distributed among Sphingobacterium, Myroides, Enterococcus, Comamonas and unclassified Flavobacteriaceae. The genus Sphingobacterium was highly prevalent among ARB from swine manure and manure-amended soil, and was also the most dominant genus among MARB from chicken manure and manure-amended soil. Other dominant genera among ARB or MARB populations in manure samples, including Myroides, Enterococcus and Comamonas, could not be detected or were detected at very low relative abundance in manure-amended soil. The present study suggests the possibility of transfer of ARBs from livestock manures to soils and persistence of ARB in these environments.

Lipid rafts control human melanoma cell migration by regulating focal adhesion disassembly.

Tumor cell migration is a crucial step in the metastatic cascade, and interruption of this step is considered to be logically effective in preventing tumor metastasis. Lipid rafts, distinct liquid ordered plasma membrane microdomains, have been shown to influence cancer cell migration, but the underlying mechanisms are still not well understood. Here, we report that lipid rafts regulate the dynamics of actin cytoskeleton and focal adhesion in human melanoma cell migration. Disrupting the integrity of lipid rafts with methyl-ß cyclodextrin enhances actin stress fiber formation and inhibits focal adhesion disassembly, accompanied with alterations in cell morphology. Furthermore, actin cytoskeleton, rather than microtubules, mediates the lipid raft-dependent focal adhesion disassembly by regulating the dephosphorylation of focal adhesion proteins and the internalization of ß3 integrin. We also show that Src-RhoA-Rho kinase signaling pathway is responsible for lipid raft disruption-induced stress fiber formation. Taken together, these observations provide a new mechanism to further explain how lipid rafts regulate the migration of melanoma cell and suggest that lipid rafts may be novel and attractive targets for cancer therapy.

Conserved electrostatic fields at the Ras-effector interface measured through vibrational Stark effect spectroscopy explain the difference in tilt angle in the Ras binding domains of Raf and RalGDS.

Vibrational Stark effect (VSE) spectroscopy was used to measure the electrostatic fields present at the interface of the human guanosine triphosphatase (GTPase) Ras docked with the Ras binding domain (RBD) of the protein kinase Raf. Nine amino acids located on the surface of Raf were selected for labeling with a nitrile vibrational probe. Eight of the probe locations were situated along the interface of Ras and Raf, and one probe was 2 nm away on the opposite side of Raf. Vibrational frequencies of the nine Raf nitrile probes were compared both in the monomeric, solvated protein and when docked with wild-type (WT) Ras to construct a comprehensive VSE map of the Ras-Raf interface. Molecular dynamics (MD) simulations employing an umbrella sampling strategy were used to generate a Boltzmann-weighted ensemble of nitrile positions in both the monomeric and docked complexes to determine the effect that docking has on probe location and orientation and to aid in the interpretation of VSE results. These results were compared to an identical study that was previously conducted on nine nitrile probes on the RBD of Ral guanidine dissociation stimulator (RalGDS) to make comparisons between the docked complexes formed when either of the two effectors bind to WT Ras. This comparison finds that there are three regions of conserved electrostatic fields that are formed upon docking of WT Ras with both downstream effectors. Conservation of this pattern in the docked complex then results in different binding orientations observed in otherwise structurally similar proteins. This work supports an electrostatic cause of the known binding tilt angle between the Ras-Raf and Ras-RalGDS complexes.

Development of a turn-on fluorescent probe for the imaging of intracellular hypochlorous acid (HClO) during ferroptosis.

Ferroptosis is a burgeoning iron-dependent cell death form, and has close relation with hypochlorous acid (HClO). Exploring the fluctuation of the HClO level in living cells during ferroptosis could contribute to the profound study of the biological functions of HClO during ferroptosis. Here, we present a turn-on probe (RH-C) for the imaging of intracellular HClO during ferroptosis. The probe RH-C utilized the N,N-dimethylthiocarbamate group as a selective recognition site for HClO, and displayed desirable sensitivity and selectivity to HClO. The probe RH-C could detect the exogenous and endogenous HClO in living cells. Furthermore, RH-C was competent in monitoring the changes of endogenous HClO level during the process of ferroptosis. Biological imaging results suggested that erastin-induced ferroptosis can result in the excessive production of the endogenous HClO, and ferrostatin-1 (Fer-1) and vitamin E (VE) could block the massive accumulation of HClO in living cells.

PLEKHM2 deficiency induces impaired mitochondrial clearance and elevated ROS levels in human iPSC-derived cardiomyocytes.

Pleckstrin homology domain-containing family M member 2 (PLEKHM2) is an essential adaptor for lysosomal trafficking and its homozygous truncation have been reported to cause early onset dilated cardiomyopathy (DCM). However, the molecular mechanism of PLEKHM2 deficiency in DCM pathogenesis and progression is poorly understood. Here, we generated an in vitro model of PLEKHM2 knockout (KO) induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to elucidate the potential pathogenic mechanism of PLEKHM2-deficient cardiomyopathy. PLEKHM2-KO hiPSC-CMs developed disease phenotypes with reduced contractility and impaired calcium handling. Subsequent RNA sequencing (RNA-seq) analysis revealed altered expression of genes involved in mitochondrial function, autophagy and apoptosis in PLEKHM2-KO hiPSC-CMs. Further molecular experiments confirmed PLEKHM2 deficiency impaired autophagy and resulted in accumulation of damaged mitochondria, which triggered increased reactive oxygen species (ROS) levels and decreased mitochondrial membrane potential (Δψm). Importantly, the elevated ROS levels caused oxidative stress-induced damage to nearby healthy mitochondria, resulting in extensive Δψm destabilization, and ultimately leading to impaired mitochondrial function and myocardial contractility. Moreover, ROS inhibition attenuated oxidative stress-induced mitochondrial damage, thereby partially rescued PLEKHM2 deficiency-induced disease phenotypes. Remarkably, PLEKHM2-WT overexpression restored autophagic flux and rescued mitochondrial function and myocardial contractility in PLEKHM2-KO hiPSC-CMs. Taken together, these results suggested that impaired mitochondrial clearance and increased ROS levels play important roles in PLEKHM2-deficient cardiomyopathy, and PLEKHM2-WT overexpression can improve mitochondrial function and rescue PLEKHM2-deficient cardiomyopathy.

Sulfochitosan inhibits P-selectin-mediated HL-60 leukocyte adhesion under flow conditions.

Excessive trafficking of leukocytes can lead to serious tissue injury. Here, four regioselectively sulfated chitosans were assessed as inhibitors of HL-60 leukocyte binding to P-selectin, by investigating their effect on leukocyte adhesion to CHO cells expressing human P-selectin under static and flow conditions. The results show that the sulfochitosans exhibit inhibitory activity in this general order: heparin > N-sulfated/6-O-sulfated chitosan ≥ 3-O,6-O-sulfated chitosan > 6-O-sulfated chitosan >> N-sulfated chitosan. This suggests that the sulfation of the double site in chitosan is essential for efficient inhibition of P-selectin-mediated HL-60 leukocyte adhesion and that such sulfochitosans may have potential as therapeutic agents against inflammatory disease.

Green synthesis of a chlorfenapyr chitosan nanopesticide for maize root application: Reducing environmental pollution and risks to nontarget organisms.

Chlorfenapyr (CHL) is a pyrrole insecticide with a novel structure that is used to control resistant pests. However, its weak systemic activity limits its application to crop roots. Herein, a novel CHL formulation with improved effective utilization rates and suitability for root application is developed to avoid or reduce contamination caused by pesticide spraying. Accordingly, we prepared CHL@CS/CMCS nanoparticle (NP) suspensions with a particle size of approximately 100 nm using chitosan (CS) and carboxymethyl chitosan (CMCS). These suspensions exhibited better thermal stability, adhesion, permeability and systemic activity than a CHL suspension concentrate (CHL-SC). The nanoformulation deposition rate on maize leaves after spraying was 12.28 mg/kg, significantly higher than that of CHL-SC. The nanosuspension was effectively absorbed and transported by roots after irrigation and was suitable for root application. The efficacy was 89.46-92.36 % against Spodoptera frugiperda at 7 d, 7.5-17.5 times higher than that of CHL-SC. Furthermore, the CHL@CS/CMCS nanosuspension was safer for earthworms. These results suggest that chitosan-based nanoformulations improve the efficacy, utilization efficiency and active period of CHL control, providing a new approach for CHL application, reducing pollutant dispersal and the environmental impacts of pesticide application and facilitating sustainable agricultural production.

Improved Calculations of Heavy Metal Toxicity Coefficients for Evaluating Potential Ecological Risk in Sediments Based on Seven Major Chinese Water Systems.

Several methods have been used to assess heavy metal contamination in sediments. However, an assessment that considers both composite heavy metal speciation and concentration is necessary to accurately study ecological risks. This study improved the potential ecological risk index method and calculated the toxicity coefficients of seven heavy metals: Arsenic (As), Cadmium (Cd), Chromium (Cr), Copper (Cu), Nickel (Ni), Lead (Pb), and Zinc (Zn). The newly calculated toxicity coefficients were validated by using previously published heavy metal distribution data of the Henan section of the Yellow River. The calculation procedure is based on the principle that the abundance of heavy metals in the environment and their bioavailable forms affect the toxicity of heavy metals. The toxicity coefficients for the seven heavy metals were calculated as follows: As = 10, Cd = 20, Cr = 5, Cu = 2, Ni = 5, Pb = 5, Zn = 1. Ecological risk assessment of the Henan section of the Yellow River using the improved toxicity coefficients revealed that the ecological risk of Cd and total heavy metals is higher than previous calculations, reaching the strength and moderate risk levels, respectively. The improved potential ecological risk index method is more sensitive to heavy metal pollution and thus provides a better indication of ecological risk. This is a necessary improvement to provide more accurate pollution assessments.

Nano-pesticide carrier O-Carboxymethyl chitosan is indigestible in Apis cerana cerana and affects intestinal flora.

O-Carboxymethyl chitosan nanoparticles (O-CMC-NPs), which are organic pesticide carriers, have excellent application potential. Exploring the effects of O-CMC-NPs on non-target organisms, such as Apis cerana cerana, is critical for their effective application; however, such studies are limited. This study investigated the stress response of A. cerana Fabricius after O-CMC-NPs ingestion. The administration of high O-CMC-NP concentrations enhanced the activities of antioxidant and detoxifying enzymes in A. cerana, with the activity of glutathione-S-transferase increasing by 54.43 %-64.33 % after one day. The transit of O-CMC-NPs into the A. cerana midgut resulted in their deposition and adherence to the intestinal wall, as they cluster and precipitate in acidic conditions. The population of Gillianella bacteria in the middle intestine was remarkably reduced after 6 d of administration of high O-CMC-NP concentrations. Contrastingly, the abundance of Bifidobacteria and Lactobacillus in the rectum significantly increased. These results indicate that the intake of high concentrations of O-CMC-NPs causes a stress response in A. cerana and affects the relative abundance of crucial intestinal flora, which may pose a potential risk to the colony. This implies that even nanomaterials with favorable biocompatibility should be applied reasonably within a specific range to avoid adverse effects on the environment and non-target organisms in the context of large-scale research and promotion of nanomaterials.

Novel application of biodegradable chitosan in agriculture: Using green nanopesticides to control Solenopsis invicta.

Botanical pesticides are biological pesticides that are environment friendly. However, their instability and short persistence limit their application. In this study, pH sensitive chitosan based rotenone (Rot) nanoparticles (CS/CMCS/Rot-NPs) were prepared using chitosan and carboxymethyl chitosan to take advantage of the acidic nature of the red fire ant midgut. Chitosan based nanoparticles showed photoprotective and slow sustained release effects on Rot and significantly increased the insecticidal activity of Rot against red fire ants. The 24-96hLC50 of CS/CMCS/Rot-NPs against red fire ants was 3.28-6.84 fold that of Rot. The CS/CMCS/Rot-NPs significantly reduced the venom alkaloid content of red fire ants and their living environment and weakened their survival by increasing their survival cost in the ecological environment. Nanotechnology combined with botanical pesticides can be used as a novel, safe, effective, and ecofriendly method to control red fire ants.

Becoming a Doctor During a Pandemic: Impact on Medical Student Social Identity Formation.

The COVID-19 pandemic required modifications to undergraduate medical education that likely affected medical students' social identity formation (SIF). SIF is shaped by experiences throughout the medical education continuum. This commentary explores factors potentially affecting medical student SIF during the COVID-19 pandemic focusing on students' perceptions of being part of the healthcare team, their role in medicine, and their engagement during the pandemic. Based on such considerations, we propose that educators should aim to design effective learning environments to support a full educational experience that encompasses acquiring medical knowledge and building strong social identities even during a pandemic.

Analysis of methods for quantifying yeast cell concentration in complex lignocellulosic fermentation processes.

Cell mass and viability are tightly linked to the productivity of fermentation processes. In 2nd generation lignocellulose-based media quantitative measurement of cell concentration is challenging because of particles, auto-fluorescence, and intrinsic colour and turbidity of the media. We systematically evaluated several methods for quantifying total and viable yeast cell concentrations to validate their use in lignocellulosic media. Several automated cell counting systems and stain-based viability tests had very limited applicability in such samples. In contrast, manual cell enumeration in a hemocytometer, plating and enumeration of colony forming units, qPCR, and in situ dielectric spectroscopy were further investigated. Parameter optimization to measurements in synthetic lignocellulosic media, which mimicked typical lignocellulosic fermentation conditions, resulted in statistically significant calibration models with good predictive capacity for these four methods. Manual enumeration of cells in a hemocytometer and of CFU were further validated for quantitative assessment of cell numbers in simultaneous saccharification and fermentation experiments on steam-exploded wheat straw. Furthermore, quantitative correlations could be established between these variables and in situ permittivity. In contrast, qPCR quantification suffered from inconsistent DNA extraction from the lignocellulosic slurries. Development of reliable and validated cell quantification methods and understanding their strengths and limitations in lignocellulosic contexts, will enable further development, optimization, and control of lignocellulose-based fermentation processes.