Evaluation of cyanotoxin L-BMAA effect on α-synuclein and TDP43 proteinopathy.

The complex interplay between genetic and environmental factors is considered the cause of neurodegenerative diseases including Parkinson's disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Among the environmental factors, toxins produced by cyanobacteria have received much attention due to the significant increase in cyanobacteria growth worldwide. In particular, L-BMAA toxin, produced by diverse taxa of cyanobacteria, dinoflagellates and diatoms, has been extensively correlated to neurodegeneration. The molecular mechanism of L-BMAA neurotoxicity is still cryptic and far from being understood. In this research article, we have investigated the molecular pathways altered by L-BMAA exposure in cell systems, highlighting a significant increase in specific stress pathways and an impairment in autophagic processes. Interestingly, these changes lead to the accumulation of both α-synuclein and TDP43, which are correlated with PD and ALS proteinopathy, respectively. Finally, we were able to demonstrate specific alterations of TDP43 WT or pathological mutants with respect to protein accumulation, aggregation and cytoplasmic translocation, some of the typical features of both sporadic and familial ALS.

Neurotoxic non-protein amino acids in commercially harvested Lobsters (Homarus americanus H. Milne-Edwards).

Cyanobacteria produce neurotoxic non-protein amino acids (NPAAs) that accumulate in ecosystems and food webs. American lobsters (Homarus americanus H. Milne-Edwards) are one of the most valuable seafood industries in Canada with exports valued at > $2 billion. Two previous studies have assessed the occurrence of ß-N-methylamino-L-alanine (BMAA) in a small number of lobster tissues but a complete study has not previously been undertaken. We measured NPAAs in eyeballs, brain, legs, claws, tails, and eggs of 4 lobsters per year for the 2021 and 2022 harvests. Our study included 4 male and 4 female lobsters. We detected BMAA and its isomers, N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB) and ß-aminomethyl-L-alanine (BAMA) by a fully validated reverse phase chromatography-tandem mass spectrometry method. We quantified BMAA, DAB, AEG and BAMA in all of the lobster tissues. Our quantification data varied by individual lobster, sex and collection year. Significantly more BMAA was quantified in lobsters harvested in 2021 than 2022. Interestingly, more BAMA was quantified in lobsters harvested in 2022 than 2021. Monitoring of lobster harvests for cyanobacterial neurotoxins when harmful algal bloom events occur could mitigate risks to human health.

Temporal dynamics of stress response in Halomonas elongata to NaCl shock: physiological, metabolomic, and transcriptomic insights.

BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.

Ectoine hyperproduction by engineered Halomonas bluephagenesis.

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine.

Protection against ß-N-methylamino-l-alanineꟷinduced vesicular monoamine transporter 2 inhibition by hydroxyl-containing proteinogenic amino acids.

ß-N-methylamino-l-alanine (BMAA) has been shown to inhibit vesicular monoamine transporter 2 (VMAT2), thereby preventing the uptake of monoaminergic neurotransmitters into platelet dense granules and synaptic vesicles. The inhibition is hypothesized to be through direct association of BMAA with hydroxyl groupꟷcontaining amino acid residues in VMAT2. This study evaluated whether BMAA-induced inhibition of VMAT2 could be prevented directly by co-incubation of BMAA with amino acids, and if this protection was specific for BMAA inhibition of VMAT2. l-tyrosine, and to a lesser extent l-serine, was able to prevent BMAA-induced VMAT2 inhibition in a concentration-dependent manner, whereas neither l-threonine nor amino acids without side chain hydroxyl groups could reduce this inhibition. Reserpine-induced VMAT2 inhibition was unaffected by any of the amino acids. These data support the hypothesized interaction between BMAA and hydroxyl groupꟷcontaining amino acids and suggests that this interaction might be leveraged to protect against the toxicity of BMAA.

How does the neurotoxin ß-N-methylamino-L-alanine exist in biological matrices and cause toxicity?

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been deemed as a risk factor for some neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). This possible link has been proved in some primate models and cell cultures with the appearance that BMAA exposure can cause excitotoxicity, formation of protein aggregates, and/or oxidative stress. The neurotoxin BMAA extensively exists in the environment and can be transferred through the food web to human beings. In this review, the occurrence, toxicological mechanisms, and characteristics of BMAA were comprehensively summarized, and proteins and peptides were speculated as its possible binding substances in biological matrices. It is difficult to compare the published data from previous studies due to the inconsistent analytical methods and components of BMAA. The binding characteristics of BMAA should be focused on to improve our understanding of its health risk to human health in the future.

Protective effects of ectoine on articular chondrocytes and cartilage in rats for treating osteoarthritis.

Osteoarthritis (OA) is a chronic degenerative disease that primarily includes articular cartilage destruction and inflammatory reactions, and effective treatments for this disease are still lacking. The present study aimed to explore the protective effects of ectoine, a compatible solute found in nature, on chondrocytes in rats and its possible application in OA treatment. In the in vitro studies, the morphology of the chondrocytes after trypsin digestion for 2 min and the viability of the chondrocytes at 50°C were observed after ectoine treatment. The reactive oxygen species (ROS) levels in chondrocytes pretreated with ectoine and post-stimulated with H2O2 were detected using an ROS assay. Chondrocytes were pretreated with ectoine before IL-1ß stimulation. RT‒qPCR was used to measure the mRNA levels of cyclooxygenase-2 (COX-2), metallomatrix proteinase-3, -9 (MMP-3, -9), and collagen type II alpha 1 (Col2A1). In addition, immunofluorescence was used to assess the expression of type II collagen. The in vivo effect of ectoine was evaluated in a rat OA model induced by the modified Hulth method. The findings revealed that ectoine significantly increased the trypsin tolerance of chondrocytes, maintained the viability of the chondrocytes at 50°C, and improved their resistance to oxidation. Compared with IL-1ß treatment alone, ectoine pretreatment significantly reduced COX-2, MMP-3, and MMP-9 expression and maintained type II collagen synthesis in chondrocytes. In vivo, the cartilage of ectoine-treated rats exhibited less degeneration and lower Osteoarthritis Research Society International (OARSI) scores. The results of this study suggest that ectoine exerts protective effects on chondrocytes and cartilage and can, therefore, be used as a potential therapeutic agent in the treatment of OA.

Identification of genes associated to ß -N oxalyl- L-α, ß-diaminopropionic acid and their role in mitigating salt stress in a low-neurotoxin cultivar of Lathyrus sativus.

Grass pea has the potential to become a miracle crop if the stigma attached to it as a toxic plant is ignored. In light of the following, we conducted transcriptome analyses on the high and low ODAP-containing cultivars i.e., Nirmal and Bidhan respectively in both normal and salt stress conditions. In this study, genes that work upstream and downstream to ß-ODAP have been found. Among these genes, AAO3 and ACL5 were related to ABA and polyamine biosynthesis, showing the relevance of ABA and polyamines in boosting the ß-ODAP content in Nirmal. Elevated ß-ODAP levels in salt stress-treated Bidhan may have evolved tolerance by positively regulating the expression of genes involved in phenylpropanoid and jasmonic acid biosynthesis. Although the concentration of ß-ODAP in Bidhan increased under salt stress, it was lower than in stress-treated Nirmal. Despite this, the expression of stress-related genes that work downstream to ß-ODAP was found higher in stress-treated Bidhan. This could be because stress-treated Nirmal has lower GSH, proline, and higher H2O2, resulting in the development of severe oxidative stress. Overall, our research not only identified new genes linked with ß-ODAP, but also revealed the molecular mechanism by which a low ß-ODAP-containing cultivar developed tolerance against salinity stress.

Recent advances in production and applications of ectoine, a compatible solute of industrial relevance.

Extremophilic bacteria growing in saline ecosystems are potential producers of biotechnologically important products including compatible solutes. Ectoine/hydroxyectoine are two such solutes that protect cells and associated macromolecules from osmotic, heat, cold and UV stress without interfering with cellular functions. Since ectoine is a high value product, overviewing strategies for improving yields become relevant. Screening of natural isolates, use of inexpensive substrates and response surface methodology approaches have been used to improve bioprocess parameters. In addition, genome mining exercises can aid in identifying hitherto unreported microorganisms with a potential to produce ectoine that can be exploited in the future. Application wise, ectoine has various biotechnological (protein protectant, membrane modulator, DNA protectant, cryoprotective agent, wastewater treatment) and biomedical (dermatoprotectant and in overcoming respiratory and hypersensitivity diseases) uses. The review summarizes current updates on the potential of microorganisms in the production of this industrially relevant metabolite and its varied applications.

Usage of ectoine as a cryoprotectant for cryopreservation of lactic acid bacteria.

Streptococcus thermophilus, the only Streptococcus species considered "Generally Recognized Safe", has been used widely in the food industry. This bacterium is one of the most valuable industrial lactic acid bacterial species. Due to the importance of this bacterium in industrial applications, it should be stored for a long time without losing its metabolic properties. The present study aimed to investigate the cryoprotectant effect of three compatible solutes (ectoine, trehalose, and sucrose) on bacterial cells stored at different temperatures (frozen at -80 °C or freeze-dried and subsequently stored at +4, -20, and -80 °C) for three months. The bacterial cells were tested for cell viability, bile salt tolerance, and lactic acid production before and after processing. The highest cell viability, bile salt tolerance, and lactic acid production were obtained with ectoine and under frozen (storage at -80 °C) conditions. In freeze-dried and subsequently stored at various temperatures, the best preservation was obtained at -80 °C, followed by -20 °C and +4 °C. Moreover, when ectoine's preservation potential was compared to other cryoprotectants, ectoine showed the highest preservation, followed by trehalose and sucrose. Although ectoine has a variety of qualities that have been proven, in the current work, we have shown for the first time that ectoine has cryoprotectant potential in yogurt starter cultures (S. thermophilus).

2-Homoectoine: An Additional Member of the Ectoine Family from Phyto- and Bacterioplankton Involved in Osmoadaptation.

Ectoine is a central osmolyte in marine plankton due to its excellent cytoprotective traits and its multifunctional roles supporting the survival of microalgae and bacteria under unfavorable environmental conditions. The protective effect of ectoine toward several kinds of stresses stirred interest in biotechnology, pharmacy, and other fields including cosmetics. Also, its hydroxylated derivative, 5-hydroxyectoine, exhibits functions similar to ectoine. Here we introduce a molecular networking-based approach to expand the family of ectoine derivatives from phyto- and bacterioplankton. A ZIC-HILIC separation protocol coupled with HRMS/MS-based molecular networking allowed us to identify the new ectoine derivative 1,4,5,6-tetrahydro-2-ethyl-4-pyrimidinecarboxylic acid, or 2-homoectoine (1). 1 is found in many algae including dinoflagellates, chlorophytes, and haptophytes. In axenic strains, the content of 1 is substantially lower. In accordance, we found that marine bacteria are prolific producers of the compound as well. This suggests that the microalgae with their associated microbiome have to be considered as sources of the compound. Increasing concentrations of the compound under high salinity suggest a role as a protectant against osmotic stress.

Metabolic engineering of high-salinity-induced biosynthesis of γ-aminobutyric acid improves salt-stress tolerance in a glutamic acid-overproducing mutant of an ectoine-deficient Halomonas elongata.

A moderately halophilic eubacterium, Halomonas elongata, has been used as cell factory to produce fine chemical 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), which functions as a major osmolyte protecting the cells from high-salinity stress. To explore the possibility of using H. elongata to biosynthesize other valuable osmolytes, an ectoine-deficient salt-sensitive H. elongata deletion mutant strain KA1 (ΔectABC), which only grows well in minimal medium containing up to 3% NaCl, was subjected to an adaptive mutagenesis screening in search of mutants with restored salt tolerance. Consequently, we obtained a mutant, which tolerates 6% NaCl in minimal medium by overproducing L-glutamic acid (Glu). However, this Glu-overproducing (GOP) strain has a lower tolerance level than the wild-type H. elongata, possibly because the acidity of Glu interferes with the pH homeostasis of the cell and hinders its own cellular accumulation. Enzymatic decarboxylation of Glu to γ-aminobutyric acid (GABA) by a Glu decarboxylase (GAD) could restore cellular pH homeostasis; therefore, we introduced an engineered salt-inducible HopgadBmut gene, which encodes a wide pH-range GAD mutant, into the genome of the H. elongata GOP strain. We found that the resulting H. elongata GOP-Gad strain exhibits higher salt tolerance than the GOP strain by accumulating high concentration of GABA as an osmolyte in the cell (176.94 µmol/g cell dry weight in minimal medium containing 7% NaCl). With H. elongata OUT30018 genetic background, H. elongata GOP-Gad strain can utilize biomass-derived carbon and nitrogen compounds as its sole carbon and nitrogen sources, making it a good candidate for the development of GABA-producing cell factories.IMPORTANCEWhile the wild-type moderately halophilic H. elongata can synthesize ectoine as a high-value osmolyte via the aspartic acid metabolic pathway, a mutant H. elongata GOP strain identified in this work opens doors for the biosynthesis of alternative valuable osmolytes via glutamic acid metabolic pathway. Further metabolic engineering to install a GAD system into the H. elongata GOP strain successfully created a H. elongata GOP-Gad strain, which acquired higher tolerance to salt stress by accumulating GABA as a major osmolyte. With the ability to assimilate biomass-derived carbon and nitrogen sources and thrive in high-salinity environment, the H. elongata GOP-Gad strain can be used in the development of sustainable GABA-producing cell factories.

Strategies for the biological production of ectoine by using different chassis strains.

As an amino acid derivative and a typical compatible solute, ectoine can assist microorganisms in resisting high osmotic pressure. Own to its long-term moisturizing effects, ectoine shows extensive applications in cosmetics, medicine and other fields. With the rapid development of synthetic biology and fermentation engineering, many biological strategies have been developed to improve the ectoine production and simplify the production process. Currently, the microbial fermentation has been widely used for large scaling ectoine production. Accordingly, this review will introduce the metabolic pathway for ectoine synthesis and also comprehensively evaluate both wild-type and genetically modified strains for ectoine production. Furthermore, process parameters affecting the ectoine production efficiency and adoption of low cost substrates will be evaluated. Lastly, future prospects on the improvement of ectoine production will be proposed.

Uptake of ß-N-methylamino-L-alanine (BMAA) into glutamate-specific synaptic vesicles: Exploring the validity of the excitotoxicity mechanism of BMAA.

The first mechanism of toxicity proposed for the cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) was excitotoxicity, and this was supported by numerous in vitro studies in which overactivation of both ionotropic and metabotropic glutamate receptors was reported. However, the excitotoxicity of BMAA is weak in comparison with other known excitotoxins and on par with that of glutamate, implying that to achieve sufficient synaptic concentrations of BMAA to cause classical in vivo excitotoxicity, BMAA must either accumulate in synapses to allow persistent glutamate receptor activation or it must be released in sufficiently high concentrations into synapses to cause the overexcitation. Since it has been shown that BMAA can be readily removed from synapses, release of high concentrations of BMAA into synapses must be shown to confirm its role as an excitotoxin in in vivo systems. This study therefore sought to evaluate the uptake of BMAA into synaptic vesicles and to determine if BMAA affects the uptake of glutamate into synaptic vesicles. There was no evidence to support uptake of BMAA into glutamate-specific synaptic vesicles but there was some indication that BMAA may affect the uptake of glutamate into synaptic vesicles. The uptake of BMAA into synaptic vesicles isolated from areas other than the cerebral cortex should be investigated before definite conclusions can be drawn about the role of BMAA as an excitotoxin.

The synthesis of ectoine enhance the assimilation of ammonia nitrogen in hypersaline wastewater by the salt-tolerant assimilation bacteria sludge.

In contrast to nitrification-denitrification microorganisms that convert ammonia nitrogen in hypersaline wastewater into nitrogen for discharge, this research utilizes sludge enriched with salt-tolerant assimilation bacteria (STAB) to assimilate organic matter and ammonia nitrogen in hypersaline wastewater into ectoine - a biomass with high economic value and resistance to external osmotic pressure. The study investigates the relationship between the synthesis of ectoine and nitrogen removal efficiency of STAB sludge in three sequencing batch reactors (SBR) operated at different salinities (50, 75, and 100 g/L) and organic matter concentrations. The research reveals that, under low concentration carbon sources (TOC/N = 4, NH4+-N = 60 mg/L), the ammonia nitrogen removal efficiency of SBR reactors increased by 14.51 % and 17.25 % within 5 d and 2 d, respectively, when salinity increased from 50 g/L to 75 g/L and 100 g/L. Under high concentration carbon sources (TOC/N = 8, NH4+-N = 60 mg/L), the ammonia nitrogen removal efficiency of STAB sludge in the three reactors stabilized at 80.20 %, 76.71 %, and 72.87 %, and the total nitrogen removal efficiency was finally stabilized at 80.47 %, 73.15 %, and 65.53 %, respectively. The nitrogen removal performance by ammonium-assimilating of STAB sludge is more sustainable under low salinity, while it is more short-term explosive under high salinity. Moreover, the intracellular ectoine concentration of STAB sludge was found to be related to this behavior. Empirical formulas confirm that STAB sludge synthesizes ectoine from nutrients in wastewater through assimilation, and intracellular ectoine has a threshold defect (150 mg/gVss). The ectoine metabolism pathways of STAB sludge was constructed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The ammonia nitrogen in sewage is converted into glutamic acid under the action of assimilation genes. It then undergoes a tricarboxylic acid cycle to synthesize the crucial precursor of ectoine - aspartic acid. Subsequently, ectoine is produced through ectoine synthase. The findings suggest that when the synthesis of intracellular ectoine reaches saturation, it inhibits the continuous nitrogen removal performance of STAB sludge under high salinity. STAB sludge does not actively release ectoine through channels under stable external osmotic pressure.

A UPLC-MS/MS method for quantification of ß-N-methylamino-L-alanine (BMAA) in Cycas sphaerica roxb. and its use in validating efficacy of a traditional BMAA removal method.

The presence of neurotoxin ß-N-Methylamino-L-alanine (BMAA) in the seeds of Cycas sphaerica is reported for first time. We developed a UPLC-MS/MS method for BMAA quantification by derivatizing with dansyl chloride. The method successfully differentiated L-BMAA from its structural isomer 2,4-diaminobutyric acid (DAB). The extracting mixture 0.1M TCA: ACN 4:1 v/v had a recovery level of >95%. The method is a high throughput sensitive chromatographic technique with 16.42 ng g-1 Limit of Quantification. BMAA was present in the endosperm of C. sphaerica, and was not detected in the leaves and pith. Washing of seeds in running cold water for 48 h reduced BMAA content by 86%. The local communities also treat the seeds under running cold water, but only for 24 h. The results of the study thus validated the traditional BMAA removal process through cold water treatment, but recommend for increase in the treatment period to 48 h or more.

Biosynthetic gene cluster profiling from North Java Sea Virgibacillus salarius reveals hidden potential metabolites.

Virgibacillus salarius 19.PP.SC1.6 is a coral symbiont isolated from Indonesia's North Java Sea; it has the ability to produce secondary metabolites that provide survival advantages and biological functions, such as ectoine, which is synthesized by an ectoine gene cluster. Apart from being an osmoprotectant for bacteria, ectoine is also known as a chemical chaperone with numerous biological activities such as maintaining protein stability, which makes ectoine in high demand in the market industry and makes it beneficial to investigate V. salarius ectoine. However, there has been no research on genome-based secondary metabolite and ectoine gene cluster characterization from Indonesian marine V. salarius. In this study, we performed a genomic analysis and ectoine identification of V. salarius. A high-quality draft genome with total size of 4.45 Mb and 4426 coding sequence (CDS) was characterized and then mapped into the Cluster of Orthologous Groups (COG) category. The genus Virgibacillus has an "open" pangenome type with total of 18 genomic islands inside the V. salarius 19.PP.SC1.6 genome. There were seven clusters of secondary metabolite-producing genes found, with a total of 80 genes classified as NRPS, PKS (type III), terpenes, and ectoine biosynthetic related genes. The ectoine gene cluster forms one operon consists of ectABC gene with 2190 bp gene cluster length, and is successfully characterized. The presence of ectoine in V. salarius was confirmed using UPLC-MS/MS operated in Multiple Reaction Monitoring (MRM) mode, which indicates that V. salarius has an intact ectoine gene clusters and is capable of producing ectoine as compatible solutes.

A Direct Analysis of ß-N-methylamino-l-alanine Enantiomers and Isomers and Its Application to Cyanobacteria and Marine Mollusks.

Of the wide variety of toxic compounds produced by cyanobacteria, the neurotoxic amino acid ß-N-methylamino-l-alanine (BMAA) has attracted attention as a result of its association with chronic human neurodegenerative diseases such as ALS and Alzheimer's. Consequently, specific detection methods are required to assess the presence of BMAA and its isomers in environmental and clinical materials, including cyanobacteria and mollusks. Although the separation of isomers such as ß-amino-N-methylalanine (BAMA), N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) from BMAA has been demonstrated during routine analysis, a further compounding factor is the potential presence of enantiomers for some of these isomers. Current analytical methods for BMAA mostly do not discriminate between enantiomers, and the chiral configuration of BMAA in cyanobacteria is still largely unexplored. To understand the potential for the occurrence of D-BMAA in cyanobacteria, a chiral UPLC-MS/MS method was developed to separate BMAA enantiomers and isomers and to determine the enantiomeric configuration of endogenous free BMAA in a marine Lyngbya mat and two mussel reference materials. After extraction, purification and derivatization with N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester ((S)-NIFE), both L- and D-BMAA were identified as free amino acids in cyanobacterial materials, whereas only L-BMAA was identified in mussel tissues. The finding of D-BMAA in biological environmental materials raises questions concerning the source and role of BMAA enantiomers in neurological disease.

Effects of the Toxic Non-Protein Amino Acid ß-Methylamino-L-Alanine (BMAA) on Intracellular Amino Acid Levels in Neuroblastoma Cells.

The cyanobacterial non-protein amino acid (AA) ß-Methylamino-L-alanine (BMAA) is considered to be a neurotoxin. BMAA caused histopathological changes in brains and spinal cords of primates consistent with some of those seen in early motor neuron disease; however, supplementation with L-serine protected against some of those changes. We examined the impact of BMAA on AA concentrations in human neuroblastoma cells in vitro. Cells were treated with 1000 µM BMAA and intracellular free AA concentrations in treated and control cells were compared at six time-points over a 48 h culture period. BMAA had a profound effect on intracellular AA levels at specific time points but in most cases, AA homeostasis was re-established in the cell. The most heavily impacted amino acid was serine which was depleted in BMAA-treated cells from 9 h onwards. Correction of serine depletion could be a factor in the observation that supplementation with L-serine protects against BMAA toxicity in vitro and in vivo. AAs that could potentially be involved in protection against BMAA-induced oxidation such as histidine, tyrosine, and phenylalanine were depleted in cells at later time points.

Ectoines production from biogas in pilot bubble column bioreactors and their subsequent extraction via bio-milking.

Despite the potential of biogas from waste/wastewater treatment as a renewable energy source, the presence of pollutants and the rapid decrease in the levelized cost of solar and wind power constrain the use of biogas for energy generation. Biogas conversion into ectoine, one of the most valuable bioproducts (1000 €/kg), constitutes a new strategy to promote a competitive biogas market. The potential for a stand-alone 20 L bubble column bioreactor operating at 6% NaCl and two 10 L interconnected bioreactors (at 0 and 6% NaCl, respectively) for ectoine production from biogas was comparatively assessed. The stand-alone reactor supported the best process performance due to its highest robustness and efficiency for ectoine accumulation (20-52 mgectoine/gVSS) and CH4 degradation (up to 84%). The increase in N availability and internal gas recirculation did not enhance ectoine synthesis. However, a 2-fold increase in the internal gas recirculation resulted in an approximately 1.3-fold increase in CH4 removal efficiency. Finally, the recovery of ectoine through bacterial bio-milking resulted in efficiencies of >70% without any negative impact of methanotrophic cell recycling to the bioreactors on CH4 biodegradation or ectoine synthesis.