Enhanced biomethane production from Scenedesmus sp. using polymer harvesting and expired COVID-19 disinfectant for pretreatment.

This study evaluates the repurposing of expired isopropanol (IPA) COVID-19 disinfectant (64% w/w) to pretreat algal biomass for enhancing methane (CH4) yield. The impact of harvesting methods (centrifugation and polymer flocculation) and microwave pretreatment on CH4 production from Scenedesmus sp. microalgal biomass were also investigated. Results show minimal impact of harvesting methods on the CH4 yield, with wet centrifuged and polymer-harvested biomass exhibiting comparable and low CH4 production at 66 and 74 L/kgvolatile solid, respectively. However, microalgae drying significantly increased CH4 yield compared to wet biomass, attributed to cell shrinkage and enhanced digestibility. Consequently, microwave and IPA pretreatment significantly enhanced CH4 production when applied to dried microalgae, yielding a 135% and 212% increase, respectively, compared to non-pretreated wet biomass. These findings underscore the advantage of using dried Scenedesmus sp. over wet biomass and highlight the synergistic effect of combining oven drying with IPA treatment to boost CH4 production whilst reducing COVID-19 waste.

Mussel-inspired resilient hydrogels with strong skin adhesion and high-sensitivity for wearable device.

Stretchable and self-adhesive conductive hydrogels hold significant importance across a wide spectrum of applications, including human-machine interfaces, wearable devices, and soft robotics. However, integrating multiple properties, such as high stretchability, strong interfacial adhesion, self-healing capability, and sensitivity, into a single material poses significant technical challenges. Herein, we present a multifunctional conductive hydrogel based on poly(acrylic acid) (PAA), dopamine-functionalized pectin (PT-DA), polydopamine-coated reduction graphene oxide (rGO-PDA), and Fe3+ as an ionic cross-linker. This hydrogel exhibits a combination of high stretchability (2000%), rapid self-healing (~ 94% recovery in 5 s), and robust self-adhesion to various substrates. Notably, the hydrogel demonstrates a remarkable skin adhesion strength of 85 kPa, surpassing previous skin adhesive hydrogels. Furthermore, incorporating rGO within the hydrogel network creates electric pathways, ensuring excellent conductivity (0.56 S m-1). Consequently, these conductive hydrogels exhibit strain-sensing properties with a significant increase in gauge factor (GF) of 14.6, covering an extensive detection range of ~ 1000%, fast response (198 ms) and exceptional cycle stability. These multifunctional hydrogels can be seamlessly integrated into motion detection sensors capable of distinguishing between various strong or subtle movements of the human body.

Metabolic response to a heterologous poly-3-hydroxybutyrate (PHB) pathway in Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host or influence transgene expression. In this study, we expressed the heterologous poly-3-hydroxybutyrate (PHB) pathway using episomal expression, which draws on the precursor acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios, as well as on alterations in AcCoA flux within the host cell. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferably shunted into the endogenous pathway for lipid biosynthesis over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA. KEY POINTS: • PHB expression had minimal effects on transcription of adjacent pathways. • N limitation favoured native lipid rather than transgenic PHB synthesis. • Glycerol addition allowed simultaneous lipid and PHB accumulation.

Occurrence, spatiotemporal trends, fate, and treatment technologies for microplastics and organic contaminants in biosolids: A review.

This review provides a comprehensive overview of the occurrence, fate, treatment and multi-criteria analysis of microplastics (MPs) and organic contaminants (OCs) in biosolids. A meta-analysis was complementarily analysed through the literature to map out the occurrence and fate of MPs and 10 different groups of OCs. The data demonstrate that MPs (54.7% occurrence rate) and linear alkylbenzene sulfonate surfactants (44.2% occurrence rate) account for the highest prevalence of contaminants in biosolids. In turn, dioxin, polychlorinated biphenyls (PCBs) and phosphorus flame retardants (PFRs) have the lowest rates (<0.01%). The occurrence of several OCs (e.g., dioxin, per- and polyfluoroalkyl substances, polycyclic aromatic hydrocarbons, pharmaceutical and personal care products, ultraviolet filters, phosphate flame retardants) in Europe appear at higher rates than in Asia and the Americas. However, MP concentrations in biosolids from Australia are reported to be 10 times higher than in America and Europe, which required more measurement data for in-depth analysis. Amongst the OC groups, brominated flame retardants exhibited exceptional sorption to biosolids with partitioning coefficients (log Kd) higher than 4. To remove these contaminants from biosolids, a wide range of technologies have been developed. Our multicriteria analysis shows that anaerobic digestion is the most mature and practical. Thermal treatment is a viable option; however, it still requires additional improvements in infrastructure, legislation, and public acceptance.

Coral endosymbiont growth is enhanced by metabolic interactions with bacteria.

Bacteria are key contributors to microalgae resource acquisition, competitive performance, and functional diversity, but their potential metabolic interactions with coral microalgal endosymbionts (Symbiodiniaceae) have been largely overlooked. Here, we show that altering the bacterial composition of two widespread Symbiodiniaceae species, during their free-living stage, results in a significant shift in their cellular metabolism. Indeed, the abundance of monosaccharides and the key phytohormone indole-3-acetic acid (IAA) were correlated with the presence of specific bacteria, including members of the Labrenzia (Roseibium) and Marinobacter genera. Single-cell stable isotope tracking revealed that these two bacterial genera are involved in reciprocal exchanges of carbon and nitrogen with Symbiodiniaceae. We identified the provision of IAA by Labrenzia and Marinobacter, and this metabolite caused a significant growth enhancement of Symbiodiniaceae. By unravelling these interkingdom interactions, our work demonstrates how specific bacterial associates fundamentally govern Symbiodiniaceae fitness.

Temperature mapping of non-photochemical quenching in Chlorella vulgaris.

Light intensity and temperature independently impact all parts of the photosynthetic machinery in plants and algae. Yet to date, the vast majority of pulse amplitude modulated (PAM) chlorophyll a fluorescence measurements have been performed at well-defined light intensities, but rarely at well-defined temperatures. In this work, we show that PAM measurements performed at various temperatures produce vastly different results in the chlorophyte Chlorella vulgaris. Using a recently developed Phenoplate technique to map quantum yield of Photosystem II (Y(II)) and non-photochemical quenching (NPQ) as a function of temperature, we show that the fast-relaxing NPQ follows an inverse normal distribution with respect to temperature and appears insensitive to previous temperature acclimation. The slow-relaxing or residual NPQ after 5 minutes of dark recovery follows a normal distribution similar to Y(II) but with a peak in the higher temperature range. Surprisingly, higher slow- and fast-relaxing NPQ values were observed in high-light relative to low-light acclimated cultures. Y(II) values peaked at the adaptation temperature regardless of temperature or light acclimation. Our novel findings show the complete temperature working spectrum of Y(II) and how excess energy quenching is managed across a wide range of temperatures in the model microalgal species C. vulgaris. Finally, we draw attention to the fact that the effect of the temperature component in PAM measurements has been wildly underestimated, and results from experiments at room temperature can be misleading.

Enhancement of cyanobacterial PHB production using random chemical mutagenesis with detection through FACS.

Poly-hydroxy-butyrate (PHB) bioplastic resin can be made directly from atmospheric CO2 using cyanobacteria. However, higher PHB productivities are required before large-scale production is economically viable. Random mutagenesis offers a way to create new production strains with increased PHB yields and increased biomass densities without complex technical manipulation associated with genetically modified organisms. This study used staining with lipid fluorescent dye (BODIPY 493/593) and fluorescence-activated cell sorting (FACS) to select high lipid content mutants and followed this with a well plate growth screen. Thirteen mutants were selected for flask cultivation and two strains produced significantly higher PHB yields (29% and 26% higher than wild type), biomass accumulation (36% and 33% higher than wild type) and volumetric PHB density (75% and 67% higher than wild type). The maximum PHB yielding strain (% dcw) was 12.0%, which was 43% higher than the wild type (8.3% in this study). The highest volumetric PHB density was 18.8 mg PHB/L compared to 10.7 mg PHB/L by the wild type. To develop cyanobacterial strain with higher PHB productivities, the combination of random chemical mutagenesis and FACS holds great potential to promote cyanobacteria bioplastic production becoming economically viable.

Comparison between human liver microsomes and the fungus Cunninghamella elegans for biotransformation of the synthetic cannabinoid JWH-424 having a bromo-naphthyl moiety analysed by high-resolution mass spectrometry.

PURPOSE: JWH-424, (8-bromo-1-naphthyl)(1-pentyl-1H-indol-3-yl)methanone, is a synthetic cannabinoid, which is a brominated analogue of JWH-018, one of the best-known synthetic cannabinoids. Despite the structural similarity to JWH-018, little is known about JWH-424 including its metabolism. The aim of the study was to compare human liver microsomes (HLM) and the fungus Cunninghamella elegans as the metabolism catalysts for JWH-424 to better understand the characteristic actions of the fungus in the synthetic cannabinoid metabolism. METHODS: JWH-424 was incubated with HLM for 1 h and Cunninghamella elegans for up to 72 h. The HLM incubation mixtures were diluted with methanol and fungal incubation mixtures were extracted with dichloromethane and reconstituted in methanol before analyses by liquid chromatography-high-resolution mass spectrometry (LC-HRMS). RESULTS: HLM incubation resulted in production of ten metabolites through dihydrodiol formation, hydroxylation, and/or ipso substitution of the bromine with a hydroxy group. Fungal incubation led to production of 23 metabolites through carboxylation, dihydrodiol formation, hydroxylation, ketone formation, glucosidation and/or sulfation. CONCLUSIONS: Generally, HLM models give good predictions of human metabolites and structural analogues are metabolised in a similar fashion. However, major hydroxy metabolites produced by HLM were those hydroxylated at naphthalene instead of pentyl moiety, the major site of hydroxylation for JWH-018. Fungal metabolites, on the other hand, had undergone hydroxylation mainly at pentyl moiety. The metabolic disagreement suggests the necessity to verify the human metabolites in authentic urine samples, while H9 and H10 (hydroxynaphthalene), H8 (ipso substitution), F22 (hydroxypentyl), and F17 (dihydroxypentyl) are recommended for monitoring of JWH-424 in urinalysis.

Effects of harvesting on morphological and biochemical characteristics of microalgal biomass harvested by polyacrylamide addition, pH-induced flocculation, and centrifugation.

The effects of microalgae harvesting methods on microalgal biomass quality were evaluated using three species namely the freshwater green alga Chlorella vulgaris, marine red alga Porphyridium purpureum and marine diatom Phaeodactylum tricornutum. Harvesting efficiencies of polyacrylamide addition, alkaline addition, and centrifugation ranged from 85 to 95, 59-92 and 100%, respectively, across these species. Morphology of the harvested cells (i.e. compromised cell walls) was significantly impacted by alkaline pH-induced flocculation for all three species. Over 50% of C. vulgaris cells were compromised with alkaline pH compared to < 10% with polyacrylamide and centrifugation. The metabolic profiles varied depending on harvesting methods. Species-specific decrease of certain metabolites was observed. These results suggest that the method of harvest can alter the metabolic profile of the biomass amongst the three harvesting methods, polyacrylamide addition showed higher harvesting efficiency with less compromised cells and higher retention of industry important biochemicals.

Corrigendum to: Investigating the impact of light quality on macromolecular of Chaetoceros muelleri.

Diatoms (Bacillariophyceae) are important to primary productivity of aquatic ecosystems. This algal group is also a valuable source of high value compounds that are utilised as aquaculture feed. The productivity of diatoms is strongly driven by light and CO2 availability, and macro- and micronutrient concentrations. The light dependency of biomass productivity and metabolite composition is well researched in diatoms, but information on the impact of light quality, particularly the productivity return on energy invested when using different monochromatic light sources, remains scarce. In this work, the productivity return on energy invested of improving growth rate, photosynthetic activity, and metabolite productivity of the diatom Chaetoceros muelleri under defined wavelengths (blue, red, and green) as well as while light is analysed. By adjusting the different light qualities to equal photosynthetically utilisable radiation, it was found that the growth rate and photosynthetic oxygen evolution was unchanged under white, blue, and green light, but it was lower under red light. Blue light improved the productivity return on energy invested for biomass, total protein, total lipid, total carbohydrate, and in fatty acids production, which would suggest that blue light should be used for aquaculture feed production.

A Cyanobacteria Enriched Layer of Shark Bay Stromatolites Reveals a New Acaryochloris Strain Living in Near Infrared Light.

The genus Acaryochloris is unique among phototrophic organisms due to the dominance of chlorophyll d in its photosynthetic reaction centres and light-harvesting proteins. This allows Acaryochloris to capture light energy for photosynthesis over an extended spectrum of up to ~760 nm in the near infra-red (NIR) spectrum. Acaryochloris sp. has been reported in a variety of ecological niches, ranging from polar to tropical shallow aquatic sites. Here, we report a new Acarychloris strain isolated from an NIR-enriched stratified microbial layer 4-6 mm under the surface of stromatolite mats located in the Hamelin Pool of Shark Bay, Western Australia. Pigment analysis by spectrometry/fluorometry, flow cytometry and spectral confocal microscopy identifies unique patterns in pigment content that likely reflect niche adaption. For example, unlike the original A. marina species (type strain MBIC11017), this new strain, Acarychloris LARK001, shows little change in the chlorophyll d/a ratio in response to changes in light wavelength, displays a different Fv/Fm response and lacks detectable levels of phycocyanin. Indeed, 16S rRNA analysis supports the identity of the A. marina LARK001 strain as close to but distinct from from the A. marina HICR111A strain first isolated from Heron Island and previously found on the Great Barrier Reef under coral rubble on the reef flat. Taken together, A. marina LARK001 is a new cyanobacterial strain adapted to the stromatolite mats in Shark Bay.

Unassembled cell wall proteins form aggregates in the extracellular space of Chlamydomonas reinhardtii strain UVM4.

The green microalga Chlamydomonas reinhardtii is emerging as a promising cell biofactory for secreted recombinant protein (RP) production. In recent years, the generation of the broadly used cell wall-deficient mutant strain UVM4 has allowed for a drastic increase in secreted RP yields. However, purification of secreted RPs from the extracellular space of C. reinhardtii strain UVM4 is challenging. Previous studies suggest that secreted RPs are trapped in a matrix of cell wall protein aggregates populating the secretome of strain UVM4, making it difficult to isolate and purify the RPs. To better understand the nature and behaviour of these extracellular protein aggregates, we analysed and compared the extracellular proteome of the strain UVM4 to its cell-walled ancestor, C. reinhardtii strain 137c. When grown under the same conditions, strain UVM4 produced a unique extracellular proteomic profile, including a higher abundance of secreted cell wall glycoproteins. Further characterization of high molecular weight extracellular protein aggregates in strain UVM4 revealed that they are largely comprised of pherophorins, a specific class of cell wall glycoproteins. Our results offer important new insights into the extracellular space of strain UVM4, including strain-specific secreted cell wall proteins and the composition of the aggregates possibly related to impaired RP purification. The discovery of pherophorins as a major component of extracellular protein aggregates will inform future strategies to remove or prevent aggregate formation, enhance purification of secreted RPs, and improve yields of recombinant biopharmaceuticals in this emerging cell biofactory. KEY POINTS: • Extracellular protein aggregates hinder purification of recombinant proteins in C. reinhardtii • Unassembled cell wall pherophorins are major components of extracellular protein aggregates • Known aggregate composition informs future strategies for recombinant protein purification.

Investigating the impact of light quality on macromolecular composition of Chaetoceros muelleri.

Diatoms (Bacillariophyceae) are important to primary productivity of aquatic ecosystems. This algal group is also a valuable source of high value compounds that are utilised as aquaculture feed. The productivity of diatoms is strongly driven by light and CO2 availability, and macro- and micronutrient concentrations. The light dependency of biomass productivity and metabolite composition is well researched in diatoms, but information on the impact of light quality, particularly the productivity return on energy invested when using different monochromatic light sources, remains scarce. In this work, the productivity return on energy invested of improving growth rate, photosynthetic activity, and metabolite productivity of the diatom Chaetoceros muelleri under defined wavelengths (blue, red, and green) as well as while light is analysed. By adjusting the different light qualities to equal photosynthetically utilisable radiation, it was found that the growth rate and photosynthetic oxygen evolution was unchanged under white, blue, and green light, but it was lower under red light. Blue light improved the productivity return on energy invested for biomass, total protein, total lipid, total carbohydrate, and in fatty acids production, which would suggest that blue light should be used for aquaculture feed production.

Cerebrospinal fluid metabolomics: detection of neuroinflammation in human central nervous system disease.

The high morbidity and mortality of neuroinflammatory diseases drives significant interest in understanding the underlying mechanisms involved in the innate and adaptive immune response of the central nervous system (CNS). Diagnostic biomarkers are important to define treatable neuroinflammation. Metabolomics is a rapidly evolving research area offering novel insights into metabolic pathways, and elucidation of reliable metabolites as biomarkers for diseases. This review focuses on the emerging literature regarding the detection of neuroinflammation using cerebrospinal fluid (CSF) metabolomics in human cohort studies. Studies of classic neuroinflammatory disorders such as encephalitis, CNS infection and multiple sclerosis confirm the utility of CSF metabolomics. Additionally, studies in neurodegeneration and neuropsychiatry support the emerging potential of CSF metabolomics to detect neuroinflammation in common CNS diseases such as Alzheimer's disease and depression. We demonstrate metabolites in the tryptophan-kynurenine pathway, nitric oxide pathway, neopterin and major lipid species show moderately consistent ability to differentiate patients with neuroinflammation from controls. Integration of CSF metabolomics into clinical practice is warranted to improve recognition and treatment of neuroinflammation.

Methyl Jasmonate and Methyl-ß-Cyclodextrin Individually Boost Triterpenoid Biosynthesis in Chlamydomonas Reinhardtii UVM4.

The commercialisation of valuable plant triterpenoids faces major challenges, including low abundance in natural hosts and costly downstream purification procedures. Endeavours to produce these compounds at industrial scale using microbial systems are gaining attention. Here, we report on a strategy to enrich the biomass of the biotechnologically-relevant Chlamydomonas reinhardtii strain UVM4 with valuable triterpenes, such as squalene and (S)-2,3-epoxysqualene. C. reinhardtii UVM4 was subjected to the elicitor compounds methyl jasmonate (MeJA) and methyl-ß-cyclodextrine (MßCD) to increase triterpene yields. MeJA treatment triggered oxidative stress, arrested growth, and altered the photosynthetic activity of the cells, while increasing squalene, (S)-2,3-epoxysqualene, and cycloartenol contents. Applying MßCD to cultures of C. reinhardtii lead to the sequestration of the two main sterols (ergosterol and 7-dehydroporiferasterol) into the growth medium and the intracellular accumulation of the intermediate cycloartenol, without compromising cell growth. When MßCD was applied in combination with MeJA, it counteracted the negative effects of MeJA on cell growth and physiology, but no synergistic effect on triterpene yield was observed. Together, our findings provide strategies for the triterpene enrichment of microalgal biomass and medium.

A comprehensive analysis of an effective flocculation method for high quality microalgal biomass harvesting.

Flocculation is a low-cost harvesting technique for microalgae biomass production, but flocculation efficiency is species dependent. In this study, we investigated the efficacy of two synthetic (polyacrylamide) and one natural (chitosan) flocculants against three algal species: the cyanobacterium Synechocystis sp., the freshwater Chlorella vulgaris, and the marine Phaeodactylum tricornutum at laboratory and pilot scales to evaluate harvesting efficiency, biomass integrity and media recycling. Growth phase affected the harvesting efficiency of the eukaryotic microalgae. The flocculation was optimal at stationary phase with high flocculation efficiency achieved using polyacrylamides at 24-36 mg/g dry weight. The effect of the flocculants on the harvested biomass was investigated. The flocculated Synechocystis sp. showed a higher proportion of compromised cells compared to C. vulgaris and P. tricornutum likely due to differences in cell walls composition. Compromised cells could lead to the release of valuable products into the surrounding growth media during flocculation. The residual culture media was recycled once with no impact on cell growth for all treatments and algal species. The flocculation technique was demonstrated at pilot-scale using 350 L microalgal suspension, showing an efficiency of 82-90% at a polyacrylamide dosage of 6.5-10 mg/L. This efficiency and the biomass quality are comparable to the laboratory-scale results. Overall, results indicate that polyacrylamide flocculants work on a wide range of species without the need for pre-treatment. The information generated in this study can contribute to making the microalgae industry more competitive.

Cerebrospinal fluid metabolites in tryptophan-kynurenine and nitric oxide pathways: biomarkers for acute neuroinflammation.

AIM: To explore the cerebrospinal fluid (CSF) metabolite features in acute neuroinflammatory diseases and identify potential biomarkers to diagnose and monitor neuroinflammation. METHOD: A cohort of 14 patients (five females, nine males; mean [median] age 7y 9mo [9y], range 6mo-13y) with acute encephalitis (acute disseminated encephalomyelitis n=6, unknown suspected viral encephalitis n=3, enteroviral encephalitis n=2, seronegative autoimmune encephalitis n=2, herpes simplex encephalitis n=1) and age-matched non-inflammatory neurological disease controls (n=14) were investigated using an untargeted metabolomics approach. CSF metabolites were analyzed with liquid chromatography coupled to high resolution mass spectrometry, followed by subsequent multivariate and univariate statistical methods. RESULTS: A total of 35 metabolites could be discriminated statistically between the groups using supervised orthogonal partial least squares discriminant analysis and analysis of variance. The tryptophan-kynurenine pathway contributed nine key metabolites. There was a statistical increase of kynurenine, quinolinic acid, and anthranilic acid in patients with encephalitis, whereas tryptophan, 3-hydroxyanthrnailic acid, and kynurenic acid were decreased. The nitric oxide pathway contributed four metabolites, with elevated asymmetric dimethylarginine and argininosuccinic acid, and decreased arginine and citrulline in patients with encephalitis. An increase in the CSF kynurenine/tryptophan ratio (p<0.001), anthranilic acid/3-hydroxyanthranilic acid ratio (p<0.001), asymmetric dimethylarginine/arginine ratio (p<0.001), and neopterin (p<0.001) strongly predicted neuroinflammation. INTERPRETATION: The combination of alterations in the tryptophan-kynurenine pathway, nitric oxide pathway, and neopterin represent a useful potential panel for neuroinflammation and holds potential for clinical translation practice. WHAT THIS PAPER ADDS: The kynurenine/tryptophan and anthranilic acid/3-hydroxyanthranilic acid ratios hold great potential as biomarkers of neuroinflammation. Elevation of the asymmetric dimethylarginine/arginine ratio in acute brain inflammation shows dysregulation of the nitric oxide pathway.

Divergence of photosynthetic strategies amongst marine diatoms.

Marine phytoplankton, and in particular diatoms, are responsible for almost half of all primary production on Earth. Diatom species thrive from polar to tropical waters and across light environments that are highly complex to relatively benign, and so have evolved highly divergent strategies for regulating light capture and utilization. It is increasingly well established that diatoms have achieved such successful ecosystem dominance by regulating excitation energy available for generating photosynthetic energy via highly flexible light harvesting strategies. However, how different light harvesting strategies and downstream pathways for oxygen production and consumption interact to balance excitation pressure remains unknown. We therefore examined the responses of three diatom taxa adapted to inherently different light climates (estuarine Thalassioisira weissflogii, coastal Thalassiosira pseudonana and oceanic Thalassiosira oceanica) during transient shifts from a moderate to high growth irradiance (85 to 1200 µmol photons m-2 s-1). Transient high light exposure caused T. weissflogii to rapidly downregulate PSII with substantial nonphotochemical quenching, protecting PSII from inactivation or damage, and obviating the need for induction of O2 consuming (light-dependent respiration, LDR) pathways. In contrast, T. oceanica retained high excitation pressure on PSII, but with little change in RCII photochemical turnover, thereby requiring moderate repair activity and greater reliance on LDR. T. pseudonana exhibited an intermediate response compared to the other two diatom species, exhibiting some downregulation and inactivation of PSII, but high repair of PSII and induction of reversible PSII nonphotochemical quenching, with some LDR. Together, these data demonstrate a range of strategies for balancing light harvesting and utilization across diatom species, which reflect their adaptation to sustain photosynthesis under environments with inherently different light regimes.

Identification of Unique 4-Methylmethcathinone (4-MMC) Degradation Markers in Putrefied Matrices†.

Drug degradation as a consequence of putrefactive bacterial activity is a well-known factor that affects the identification and quantitation of certain substances of forensic interest. Current knowledge on putrefaction-mediated degradation of drugs is, however, significantly lacking. This study aimed to investigate the degradation of 4-methylmethcathinone (4-MMC or mephedrone) and to detect its degradation products in putrefied biological matrices containing 4-MMC. The bacteria species Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris were grown in brain-heart infusion broth, spiked with 4-MMC and incubated at 37°C for 24 h. Postmortem human blood and fresh porcine liver macerate were also left to putrefy in sample tubes at room temperature for 1 week. Structural elucidation was based on modern spectroscopic analyses including the use of high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. All four putrefactive bacteria were capable of degrading 4-MMC extensively under the experimental conditions explored. Of particular interest was the discovery of a novel degradation product common to all four bacterial species, which was assigned as 2-hydroxy-1-(4-methylphenyl)propan-1-one (HMP) based on the spectroscopic data. This degradation product was detectable in both postmortem human blood and porcine liver samples. The stability of the identified degradation products, especially HMP, should be further investigated to assess their validity of serving as marker analytes for monitoring 4-MMC in postmortem toxicology.

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology.

Diatoms are photosynthetic microeukaryotes that dominate phytoplankton populations and have increasing applicability in biotechnology. Uncovering their complex biology and elevating strains to commercial standards depends heavily on robust genetic engineering tools. However, engineering microalgal genomes predominantly relies on random integration of transgenes into nuclear DNA, often resulting in detrimental "position-effects" such as transgene silencing, integration into transcriptionally-inactive regions, and endogenous sequence disruption. With the recent development of extrachromosomal transgene expression via independent episomes, it is timely to investigate both strategies at the phenotypic and genomic level. Here, we engineered the model diatom Phaeodactylum tricornutum to produce the high-value heterologous monoterpenoid geraniol, which, besides applications as fragrance and insect repellent, is a key intermediate of high-value pharmaceuticals. Using high-throughput phenotyping we confirmed the suitability of episomes for synthetic biology applications and identified superior geraniol-yielding strains following random integration. We used third generation long-read sequencing technology to generate a complete analysis of all transgene integration events including their genomic locations and arrangements associated with high-performing strains at a genome-wide scale with subchromosomal detail, never before reported in any microalga. This revealed very large, highly concatenated insertion islands, offering profound implications on diatom functional genetics and next generation genome editing technologies, and is key for developing more precise genome engineering approaches in diatoms, including possible genomic safe harbour locations to support high transgene expression for targeted integration approaches. Furthermore, we have demonstrated that exogenous DNA is not integrated inadvertently into the nuclear genome of extrachromosomal-expression clones, an important characterisation of this novel engineering approach that paves the road to synthetic biology applications.