Microbial Research Articles

Microbial Biodiversity in Sediment from the Amuyo Ponds: Three Andean Hydrothermal Lagoons in Northern Chile

The Amuyo Ponds (APs) are a group of three brackish hydrothermal lagoons located at 3700 m above sea level in a pre-Andean setting in the Atacama Desert. Each pond shows a conspicuous green (GP), red (RP), or yellow (YP) coloration, and discharges water rich in arsenic and boron into the Caritaya River (Camarones Basin, northern Chile). Microorganisms are subjected to harsh environmental conditions in these ponds, and the microbial composition and diversity in the Amuyo Ponds’ sediments are unknown. The microbial life colonizing AP sediments was explored by metagenomics analyses, showing a diverse microbial life dominated by members of the bacterial domain, with nearly 800 bacterial genome sequences, and sequences associated with Archaea, Eukarya, and viruses. The genus Pseudomonas was more abundant in GP and YP sediments, while the genera Pseudomonas, Aeromonas, and Shewanella were enriched in RP sediments. Archaeal composition was similar in all sediments, and enriched with methanogens sequences from the Archaeoglobi and Halobacteria classes. Abundant fungi sequences were detected in all sediments from the phyla Blastocladiomycota and Ascomycota. We also report putative functional capabilities related to virulence and defense genes, the biosynthesis of secondary metabolites, and tolerance to arsenic. Thirteen bacterial and fourteen viral metagenome-assembled genomes were reconstructed and informed here. This work expands our knowledge on the richness of the microorganisms in the APs and open further studies on the ecology and genomics of this striking Andean geosite.

Valorizing Waste Streams to Enhance Sustainability and Economics in Microbial Oil Production.

Driven by demand for more sustainable products, research and capital investment has been committed to developing microbially produced oils. While researchers have shown oleaginous yeasts and other microbes can produce low-carbon footprint oils by leveraging waste streams as energy sources, previous analyses have not fully explored the quantity of available waste streams and in turn economy-of-scale enabled on capital and operating expenses. This paper makes parallels to 2G ethanol facilities, enabling a data-driven understanding of large-scale production economics. Production costs are broken down for a variety of scenarios. The analysis finds that reaching price parity with large-scale commodity oils (e.g., palm oil, high-oleic cooking oils, biofuels feedstock oils, lauric acid) is not possible today and unlikely even under aggressive future assumptions about strain productivity. Instead, commercial production must be targeted at end markets where sustainability-conscious consumers are willing to pay the price premiums identified in this paper.

Design and computational analysis of a novel Azurin-BR2 chimeric protein against breast cancer

Abstract Cancer is one of most lethal diseases worldwide. Chemotherapeutics and surgeries are among the treatment facilities available for curing cancer. However due to their negative impact on normal cells and drug resistance development, new treatment strategies have yet to be developed. Some microbial products exhibit therapeutic potential for treating cancer. Pseudomonas aeruginosa Azurins have shown anticancer effects against breast cancer without affecting normal cells. To enhance its cytotoxic effect and targeted delivery, we fused Azurin with a cell-penetrating peptide (BR2) through a rigid linker and evaluated its anticancer potential via in silico analysis. The prediction of the secondary and the tertiary structures and analysis of physiochemical properties of chimeric proteins were computationally performed. The Azurin-BR2 chimeric protein has a basic nature with a molecular weight of 16.8 kDa. The quality indices and validation of chimeric proteins were performed with ERRAT2 and Ramachandran plot values, respectively. The quality index of the chimeric protein was predicted to be 81% to 84.6%, and residues residing in the most favoured region were identified. The HDOCK bioinformatics tool was used for docking a chimeric protein with a cancer suppressor protein p53. The results of the current study support that an Azurin-BR2 fusion protein has a high binding affinity for p53 can induce apoptosis in cancerous cells, and can be used in tumor-targeting therapy.

Mathematical model of dissolved microbial products in sewage treatment system

Water is the source of life, but all kinds of water resources in the world are suffering from different degrees of pollution. Water pollution leads to a serious shortage of available fresh water resources, and sewage treatment is the main way to solve water pollution. For the sewage after biological treatment of activated sludge, the organic matter contained in the effluent is mainly the dissolved microbial products (SMPs) produced in the process of microbial metabolism. The composition of SMPs is complex, mainly including macromolecular substances such as protein, polysaccharide, humic acid and DNA and cell fragments. The mathematical model of activated sludge is a quantitative description of the mathematical relationship between substrate degradation parameters and microbial growth. Starting from the Monod equation representing the relationship between substrate consumption and microbial growth, it combines the reactor theory and microbiology theory in the chemical field. Based on the principle of conservation of materials and Monod equation, the mathematical expression of organic degradation model was determined according to the collected data and empirical values of parameters. The general idea of activated sludge model No.1 (ASM1) for activated sludge process simulation was introduced, and the influence of sludge concentration on SBR process water treatment was explored. It was found that the removal rate of COD, ammonia nitrogen, total nitrogen and total phosphorus increases with the increase of sludge concentration. When C/N=8, the removal rate of ammonia nitrogen increased from 62% to 81%, and the removal rate of total nitrogen increased from 64% to 82%, with the most obvious effect.

Morphological, Biochemical and Molecular Characterization of Plant Growth Promoting Rhizobacteria with Synergistic Effect against Fusarium oxysporum

Currently, plant growth-promoting rhizosphere bacteria (PGPR) are heavily exploited as microbial inoculants in agricultural production among which Pseudomonas sp. and Bacillus sp. are widely used in plant growth promotion and disease control as excellent inoculum strains. Plant growth-promoting rhizobacteria has the potential to be used in agriculture to promote plant growth and health through various mechanisms. Therefore, identifying novel strains tailored to specific agricultural requirements continues to be a key area of research. In the present research work, the strains of PGPR isolated from soybean rhizosphere at Department of Plant Pathology and characterized at morphological, biochemical and molecular level. These strains are now used in ‘Biomix’ formulation a microbial consortium product developed at the Department of Plant Pathology VNMKV, Parbhani to benefit the farmers. Studying the molecular, biochemical, and morphological traits of these microbial strains which are now involved in VNMKV produced ‘Biomix formulation is crucial to maintain its consistency, purity and optimize its formulation. Molecular characterization and phylogenetic analysis will ensure accurate identification and classification of the isolates, enhancing the biofertilizer's effectiveness in agriculture. All three isolates have been identified as per Bergey’s Manual for the determination of bacteriology. All isolates tested positive for starch hydrolysis, citrate utilization, catalase activity, indole production, gelatine hydrolysis, and tetrazolium reduction. Additionally, the 16S rDNA gene sequences of the various potential isolates were validated as P. fluorescens VNMKV1, P. putida VNMKV1, and B. subtilis VNMKV1. The potential of these novel strains of plant growth-promoting rhizobacteria as biocontrol agents was explored by screening against soil-borne pathogenic fungi highlighting their potential to be used as bioinoculant agents.

Amplified selenite toxicity in methanogenic archaea mediated by cysteine.

The challenge of understanding the interaction between trace elements and microbial life is critical for assessing environmental and ecological impacts. Nevertheless, cysteine (Cys), a low molecular weight thiol substance prevalent in the ecosystem, is able to influence the fate of certain trace elements, which increases the complexity of the interaction between trace elements and microorganisms. Therefore, we chose Cys, selenite and the model methanogenic archaeon Methanosarcina acetivorans C2A as research targets, and comprehensively explored the intricate role of Cys in modulating the biological effects of selenite on M. acetivorans C2A in terms of population growth, methane production and oxidative stress. Our results demonstrate that Cys significantly exacerbates the inhibitory effects of selenite on growth and methane production in M. acetivorans C2A. This increased toxicity is linked to heightened membrane permeability and oxidative stress, with a marked upregulation in reactive oxygen species and changes in NADPH levels. Transcriptomic analysis reveals alterations in genes associated with transmembrane transport and methanogenesis. Intriguingly, we also observed a potential interaction between selenite and phosphate transmembrane transporters, suggesting a novel pathway for selenite entry into cells. These findings highlight the complex interplay between trace elements and microbial processes, with significant implications for understanding environmental risks and developing remediation strategies.

Effects of inorganic nitrogen enrichment on soil CH4 and CO2 production in freshwater and mesohaline marshes across six estuaries in China

Eutrophication is an important environmental stressor in coastal wetlands that alters ecosystem processes including primary production and the carbon cycle. However, how different nitrogen eutrophication scenarios may affect CH4 and CO2 production along a salinity gradient in coastal wetlands is poorly known. We collected the surface soil samples from both tidal freshwater and mesohaline Phragmites australis marshes in six main estuaries in China and conducted inorganic nitrogen enrichment anaerobic incubation experiments. Background soil CH4 and CO2 production rates were strongly correlated with total nitrogen but not salinity. On average, nitrate enrichment decreased soil CH4 and CO2 production rates by ca. 20 % in freshwater marsh and 16–43 % in mesohaline marshes, whereas ammonium enrichment decreased CO2 production by ca. 26–30 % but had no effect on CH4 production. Salinity was not an important mitigating factor in either eutrophication scenario. In most cases, nitrogen addition decreased the extracellular enzyme activities of β-1,4-glucosidase and cellobiohydrolase, but increased the activity of β-N-acetyl glucosaminidase, phenol oxidase and peroxidase. Nitrate addition decreased the mcrA gene abundance on average by 34–35 % but ammonium addition had insignificant effect. Total nitrogen availability was an important driver of CH4 and CO2 production rates in these tidally influenced coastal wetlands. Our results showed that tidal marshes with salinity < 15 ppt had similar soil CH4 production rates, and increasing inorganic nitrogen eutrophication might lower soil microbial carbon gas production in both tidal freshwater and mesohaline marshes.

Redox Gradient Shapes the Chemical Composition of Peatland Microbial Communities.

The response of soil carbon to climate change and anthropogenic forcing depends on the relationship between the physicochemical variables of the environment and microbial communities. In anoxic soils that store large amounts of organic carbon, it can be hypothesized that the low amount of catabolic energy available leads microbial organisms to minimize the energy costs of biosynthesis, which may shape the composition of microbial communities. To test this hypothesis, thermodynamic modeling was used to assess the link between redox gradients in the ombrotrophic peatland of the Marcell Experimental Forest (Minnesota, USA) and the chemical and taxonomic composition of microbial communities. The average amino acid composition of community-level proteins, called hereafter model proteins, was calculated from shotgun metagenomic sequencing. The carbon oxidation state of model proteins decreases linearly from -0.14 at 10 cm depth to -0.17 at 150 cm depth. Calculating equilibrium activities of model proteins for a wide range of chemical conditions allows identification of the redox potential of maximum chemical activity. Consistent with redox measurements across peat soils, this model Eh decreases logarithmically from an average value of 300 mV at 10 cm depth, close to the stability domain of goethite relative to Fe2+, to an average value of -200 mV at 150 cm, within the stability domain of CH4 relative to CO2. The correlation identified between the taxonomic abundance and the carbon oxidation state of model proteins enables predicting the evolution of taxonomic abundance as a function of model Eh. The model taxonomic abundance is consistent with the measured gene and taxonomic abundance, which evolves from aerobic bacteria at the surface including Acidobacteria, Proteobacteria, and Verrumicrobia, to anaerobes at depth dominated by Crenarchaeota. These results indicate that the thermodynamic forcing imposed by redox gradient across peat soils shapes both the chemical and taxonomic composition of microbial communities. By providing a mechanistic understanding of the relationship between microbial community and environmental conditions, this work sheds new light on the mechanisms that govern soil microbial life and opens up prospects for predicting geochemical and microbial evolution in changing environments.

De Novo Biosynthesis of a Bioactive Meroterpene Bakuchiol in Yeast.

Bakuchiol (BAK), a specialized meroterpene, is known for its valuable biological properties and has recently gained prominence in cosmetology for its retinol-like functionality. However, low abundance in natural sources leads to environmentally unfriendly and unsustainable practices associated with crop-based manufacturing and chemical synthesis. Here, we identified a prenyltransferase (PT) from Psoralea corylifolia that catalyzes the reverse geranylation of a nonaromatic carbon in para-coumaric acid (p-CA), coupled with a decarboxylation step to form BAK. Given that the biosynthesis pathway of BAK is well elucidated, we engineered Saccharomyces cerevisiae to produce BAK, starting from glucose. To enhance the titer of BAK, we employed a multifaceted approach that included increasing the supply of precursors, balancing the fluxes in the two parallel biosynthetic pathways, engineering of prenyltransferase, and fusing enzymes. Consequently, the engineered yeast strains showed a marked improvement of 117.3-fold in BAK production, reaching a titer of 9.28 mg/L from glucose. Our work provides a viable approach for the sustainable microbial production of complex natural meroterpenes.

Microorganisms in Fermented Foods and Their Effects on the Human Body

In recent years, microorganisms in fermented foods have shown an important role in regulating human health. Studies have shown that probiotics have significant health benefits in fermented foods, such as improving gut health, stimulating immune function, and promoting metabolism, and mental health. Specifically, lactic acid bacteria in yogurt can increase the number of beneficial bacteria in the intestines and reduce the growth of pathogenic bacteria, and yeasts such as Saccharomyces cerevae have multiple health effects on intestinal microbiota protection and anti-inflammation. In addition, various enzymes and metabolites produced by Aspergillus during fermentation process can improve the nutritional value of food. However, although lots of research has been done, the understanding of the microbial mechanism under different fermentation conditions is still insufficient. In this article, the types and influencing factors of microorganisms in yogurt, wine and seasoner were reviewed. The effects of temperature, pH value, fermentation time and other conditions on microbial activity and product quality were analyzed. The results showed that suitable fermentation conditions could significantly increase microbial activity, improve product flavor and health function, and provide a theoretical basis for optimizing the production process of fermented food. The article provides a theoretical basis for improving the fermented food production process, thus helping to improve product quality and market competitiveness. However, this article has some limitations like the lack of understanding of interactions between different microorganisms. Future research should focus on examining the collaborative relationship between these microbes to improve the health benefits and production efficiency of fermented foods.

Maternal consumption of yoghurt activating the aryl hydrocarbon receptor increases group 3 innate lymphoid cells in murine offspring

ABSTRACT Indole derivatives are microbial metabolites of the tryptophan pathway involved in gut immune homeostasis. They bind to the aryl hydrocarbon receptor (AhR), thereby modulating development of intestinal group 3 innate lymphoid cells (ILC3) and subsequent interleukin-22 production. In mice, indole derivatives of the maternal microbiota can reach the milk and drive early postnatal ILC3 development. Apart from the gut microbiota, lactic acid bacteria (LAB) also produce indole compounds during milk fermentation. Using germ-free mice, the aim of our study was to test if maternal intake of a dairy product enriched in AhR-activating indoles produced by fermentation could boost maturation of the intestinal innate immune system in the offspring. A set of 631 LAB strains were genetically screened for their potential to produce indole compounds. Among these, 125 strains were tested in combination with standard strains to produce yoghurts that were screened for their ability to activate AhR in vitro using the HepG2–AhR–Luc cell line. The most active yoghurt and a control yoghurt were formulated as pellets and fed to germ-free dams during pregnancy and lactation. Analysis of the offspring on postnatal day 14 using flow cytometry revealed an increase in the frequency of small intestinal lamina propria NKp46 +ILC3 s in the pups born to dams that had consumed the purified diet containing an AhR-active yoghurt (AhrY-diet) compared to control yoghurt (ConY-diet). Selection of LABs based on their ability to produce a fermented dairy able to activate AhR appears to be an effective approach to produce a yoghurt with immunomodulatory properties. IMPORTANCE Key progresses in the sequencing and functional annotation of microbial organisms have revolutionized research in the fields of human metabolism and food biotechnology. In particular, the gut microbiome is now recognized as an important mediator of the impact of nutrition on human metabolism. Annotated genomes of a large number of bacteria are now available worldwide, which selectively transform food through fermentation to produce specific bioactive compounds with the potential to modulate human health. A previous research has demonstrated that the maternal microbiota shapes the neonatal immune system. Similarly, this report shows that lactic acid bacteria can be selected to produce fermented food that can also modulate postnatal intestinal immunity.

Ashbya gossypii as a versatile platform to produce sabinene from agro-industrial wastes

BackgroundAshbya gossypii is a filamentous fungus widely utilized for industrial riboflavin production and has a great potential as a microbial chassis for synthesizing other valuable metabolites such as folates, biolipids, and limonene. Engineered strains of A. gossypii can effectively use various waste streams, including xylose-rich feedstocks. Notably, A. gossypii has been identified as a proficient biocatalyst for producing limonene from xylose-rich sources. This study aims to investigate the capability of engineered A. gossypii strains to produce various plant monoterpenes using agro-industrial waste as carbon sources.ResultsWe overexpressed heterologous terpene synthases to produce acyclic, monocyclic, and bicyclic monoterpenes in two genetic backgrounds of A. gossypii. These backgrounds included an NPP synthase orthogonal pathway and a mutant erg20F95W allele with reduced FPP synthase activity. Our findings demonstrate that A. gossypii can synthesize linalool, limonene, pinene, and sabinene, with terpene synthases showing differential substrate selectivity for NPP or GPP precursors. Additionally, co-overexpression of endogenous HMG1 and ERG12 with heterologous NPP synthase and terpene synthases significantly increased sabinene yields from xylose-containing media. Using mixed formulations of corn-cob lignocellulosic hydrolysates and either sugarcane or beet molasses, we achieved limonene and sabinene productions of 383 mg/L and 684.5 mg/L, respectively, the latter representing a significant improvement compared to other organisms in flask culture mode.ConclusionsEngineered A. gossypii strains serve as a suitable platform for assessing plant terpene synthase functionality and substrate selectivity in vivo, which are crucial to understand monoterpene bioproduction. The NPP synthase pathway markedly enhances limonene and sabinene production in A. gossypii, achieving levels comparable to those of other industrial microbial producers. Furthermore, these engineered strains offer a novel approach for producing monoterpenes through the valorization of agro-industrial wastes.

Investigation of Bottleneck Enzyme Through Flux Balance Analysis to Improve Glycolic Acid Production in Escherichia coli.

Amid rising environmental concerns, attempts have been made to produce glycolic acid (GA) using microbial processes with renewable carbon resources instead of using chemicals. The Dahms pathway for GA production uses xylose as a substrate and consists of relatively simple enzymatic steps. However, employing it leads to a decrease in cell growth and GA productivity. Systematically identifying and addressing metabolic bottlenecks in the Dahms pathway are essential for efficient glycolic acid (GA) production have not yet been performed. Through metabolic flux balance analysis, we found that insufficient aldehyde dehydrogenase (AldA) activity lowers GA production and negatively affects cell growth due to reduced energy production. Thus, we discovered a novel AldA isolated from Buttiauxella agrestis (BaAldA) demonstrated a 1.69-fold lower KM and a 1.49-fold higher turnover rate (kcat/KM) than AldA from Escherichia coli (EcAldA). GA production in E. coli harboring BaAldA was 1.59 times higher than in the original strain. Fed-batch fermentation of E. coli harboring BaAldA produced 22.70g/L GA with a yield of 0.497g/gxylose (98.2% of the theoretical maximum yield in the Dahms pathway), showing a higher final yield for GA than previously reported in E. coli. Our novel BaAldA enzyme shows great potential for the production of GA using microorganisms or enzymes. Furthermore, our approach to identifying metabolic bottlenecks using flux balance analysis could be utilized to enhance the microbial production of various desirable products in future studies.

Integration of plant and microbial oil processing at oilcane biorefineries for more sustainable biofuel production

AbstractOilcane—an oil‐accumulating crop engineered from sugarcane—and microbial oil have the potential to improve renewable oil production and help meet the expected demand for bioderived oleochemicals and fuels. To assess the potential synergies of processing both plant and microbial oils, the economic and environmental implications of integrating microbial oil production at oilcane and sugarcane biorefineries were characterized. Due to decreased crop yields that lead to higher simulated feedstock prices and lower biorefinery capacities, current oilcane prototypes result in higher costs and carbon intensities than microbial oil from sugarcane. To inform oilcane feedstock development, we calculated the required biomass yields (as a function of oil content) for oilcane to achieve financial parity with sugarcane. At 10 dw% oil, oilcane can sustain up to 30% less yield than sugarcane and still be more profitable in all simulated scenarios. Assuming continued improvements in microbial oil production from cane juice, achieving this target results in a minimum biodiesel selling price of 1.34 [0.90, 1.85] USD∙L−1 (presented as median [5th, 95th] percentiles), a carbon intensity of 0.51 [0.47, 0.55] kg CO2e L−1, and a total biodiesel yield of 2140 [1870, 2410] L ha−1 year−1. Compared to biofuel production from soybean, this outcome is equivalent to 3.0–3.9 as much biofuel per hectare of land and a 57%–63% reduction in carbon intensity. While only 20% of simulated scenarios fell within the market price range of biodiesel (0.45–1.11 USD∙L−1), if the oilcane biomass yield would improve to 25.6 DMT∙ha−1∙y−1 (an equivalent yield to sugarcane) 87% of evaluated scenarios would have a minimum biodiesel selling price within or below the market price range.

Real Case Study of 600 m3 Bubble Column Fermentations: Spatially Resolved Simulations Unveil Optimization Potentials for l-Phenylalanine Production With Escherichia coli.

Large-scale fermentations (»100 m³) often encounter concentration gradients which may significantly affect microbial activities and production performance. Reliably investigating such scenarios in silico would allow to optimize bioproduction. But related simulations are very rare in particular for large bubble columns. Here, we pioneer the spatially resolved investigation of a 600 m³ bubble column operating for Escherichia coli based l-phenylalanine fed-batch production. Microbial kinetics are derived from experimental data. Advanced Euler-Lagrange (EL) computational fluid dynamics (CFD) simulations are applied to track individual bubble dynamics that result from a recently developed bubble breakage model. Thereon, the complex nonlinear characteristics of hydrodynamics, mass transfer, and microbial activities are simulated for large scale and compared with real data. As a key characteristic, zones for upriser, downcomer, and circulation cells were identified that dominate mixing and mass transfer. This results in complex gradients of glucose, dissolved oxygen, and microbial rates dividing the bioreactor into sections. Consequently, alternate feed designs are evaluated splitting real feed rates in two feeds at different locations. The opposite reversed installation of feed spots and spargers improved the product synthesis by 6.24% while alternate scenarios increased the growth rate by 11.05%. The results demonstrate how sophisticated, spatially resolved simulations of hydrodynamics, mass transfer, and microbial kinetics help to optimize bioreactors in silico.

Rational and Semirational Approaches for Engineering Salicylate Production in Escherichia coli.

Salicylate plays a pivotal role as a pharmaceutical intermediate in drugs, such as aspirin and lamivudine. The low catalytic efficiency of key enzymes and the inherent toxicity of salicylates to cells pose significant challenges to large-scale microbial production. In this study, we introduced the salicylate synthase Irp9 into an l-phenylalanine-producing Escherichia coli, constructing the shortest salicylate biosynthetic pathway. Subsequent protein engineering increased the catalytic efficiency of Irp9 by 33.5%. Furthermore, by integrating adaptive evolution with transcriptome analysis, we elucidated the crucial mechanism of efflux proteins in salicylate tolerance. The elucidation of this mechanism guided us in the targeted modification of these transport proteins, achieving a reported maximum level of 3.72 g/L of salicylate in a shake flask. This study highlights the importance of efflux proteins for enhancing the productivity of microbial cell factories in salicylate production, which also holds potential for application in the green synthesis of other phenolic acids.

The Molecules Gateway: A Homogeneous, Searchable Database of 150k Annotated Molecules from Actinomycetes.

Natural products are a sustainable resource for drug discovery, but their identification in complex mixtures remains a daunting task. We present an automated pipeline that compares, harmonizes and ranks the annotations of LC-HRMS data by different tools. When applied to 7,400 extracts derived from 6,566 strains belonging to 86 actinomycete genera, it yielded 150,000 molecules after processing over 50 million MS features. The web-based Molecules Gateway provides a highly interactive access to experimental and calculated data for these molecules, along with the metadata related to extracts and producer strains. We show how the Molecules Gateway can be used to rapidly identify known hard to find microbial products, unreported analogs of known families and not yet described metabolites. The Molecules Gateway, which complements available repositories, contains annotated MS data, both acquired and computationally processed under an identical workflow, making it suitable for global analyses which reveal a large and untapped chemical diversity afforded by actinomycetes.

Recovery of ammonia assimilating microbiome after Cr (VI) shock by bio-accelerators

The pretreatment process is often unable to completely intercept heavy metals in wastewater, facing a huge risk of leakage, increasing the difficulty of treating pollutants in the subsequent biochemical process or even leading to the collapse of the system, and facing the difficulty of inoperability and rehabilitation. Heterotrophic ammonia assimilation has the potential to maintain some stability after heavy metal shock, thanks to its rapid microbial proliferation, robust resistance to high loads, remarkable environmental adaptability, and inherent stability. Bio-accelerators dosing strategies could strengthen the performance recovery ability of traditional bio-system after heavy metal impact. However, no recovery strategies for inhibiting HAA have been reported. Herein, three bio-accelerants, specifically, vitamin A, 6-benzylaminopurine, and α-ketoglutaric acid, were investigated for their potential to restore the HAA system impacted by 20 mg/L Cr (VI). The three bio-accelerants effectively mitigated the toxicity of the HAA system, resulting in a 60.4% increase in NH4+-N removal efficiency within just 6 days with cytokinin. During toxicity remediation, three bio-accelerants facilitated the production of extracellular protein components in soluble microbial products and stimulated the secretion of extracellular polymeric substances. The three bio-accelerants enhanced competition among genera and influenced community assembly processes to regulate community structure and enhance functional gene expression. This study offers a practical approach to enhancing the HAA process and remediating microbial toxicity.

Isotopic evidence of acetate turnover in Precambrian continental fracture fluids

The deep continental crust represents a vast potential habitat for microbial life where its activity remains poorly constrained. Organic acids like acetate are common in these ecosystems, but their role in the subsurface carbon cycle - including the mechanism and rate of their turnover - is still unclear. Here, we develop an isotope-exchange ‘clock’ based on the abiotic equilibration of H-isotopes between acetate and water, which can be used to define the maximum in situ acetate residence time. We apply this technique to the fracture fluids in Birchtree and Kidd Creek mines within the Canadian Precambrian crust. At both sites, we find that acetate residence times are <1 million years and calculated a rate of turnover that could theoretically support microbial life. However, radiolytic water-rock reactions could also contribute to acetate production and degradation, a process that would have global relevance for the deep biosphere. More broadly, our study demonstrates the utility of isotope-exchange clocks in determining residence times of biomolecules with possible applications to other environments.

Engineering Yarrowia lipolytica to Produce l-Malic Acid from Glycerol.

The declining availability of cheap fossil-based resources has sparked growing interest in the sustainable biosynthesis of organic acids. l-Malic acid, a crucial four-carbon dicarboxylic acid, finds extensive applications in the food, chemical, and pharmaceutical industries. Synthetic biology and metabolic engineering have enabled the efficient microbial production of l-malic acid, albeit not in Yarrowia lipolytica, an important industrial microorganism. The present study aimed to explore the potential of this fungal species for the production of l-malic acid. First, endogenous biosynthetic genes and heterologous transporter genes were overexpressed in Y. lipolytica to identify bottlenecks in the l-malic acid biosynthesis pathway grown on glycerol. Second, overexpression of isocitrate lyase, malate synthase, and malate dehydrogenase in the glyoxylate cycle pathway and introduction of a malate transporter from Schizosaccharomyces pombe significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent increase to 37.0 g/L was attained through shake flask medium optimization. Third, adaptive laboratory evolution allowed the engineered strain Po1g-CEE2+Sp to tolerate a lower pH and to accumulate a higher amount of l-malic acid (56.0 g/L). Finally, when scaling up to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion, this study demonstrates for the first time the successful production of l-malic acid in Y. lipolytica by combining metabolic engineering and laboratory evolution, paving the way for large-scale sustainable biosynthesis of this and other organic acids.