Nitrogen Sources Reprogram Carbon and Nitrogen Metabolism to Promote Andrographolide Biosynthesis in Andrographis paniculata (Burm.f.) Nees Seedlings.

Carbon (C) and nitrogen (N) metabolisms participate in N source-regulated secondary metabolism in medicinal plants, but the specific mechanisms involved remain to be investigated. By using nitrate (NN), ammonium (AN), urea (UN), and glycine (GN), respectively, as sole N sources, we found that N sources remarkably affected the contents of diterpenoid lactone components along with C and N metabolisms reprograming in Andrographis paniculata, as compared to NN, the other three N sources raised the levels of 14-deoxyandrographolide, andrographolide, dehydroandrographolide (except UN), and neoandrographolide (except AN) with a prominent accumulation of farnesyl pyrophosphate (FPP). These N sources also raised the photosynthetic rate and the levels of fructose and/or sucrose but reduced the activities of phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoenolpyruvate carboxylase (PEPC) and pyruvate dehydrogenase (PDH). Conversely, phosphoenolpyruvate carboxykinase (PEPCK) and malate enzyme (ME) activities were upregulated. Simultaneously, citrate, cis-aconitate and isocitrate levels declined, and N assimilation was inhibited. These results indicated that AN, UN and GN reduced the metabolic flow of carbohydrates from glycolysis into the TCA cycle and downstream N assimilation. Furthermore, they enhanced arginine and GABA metabolism, which increased C replenishment of the TCA cycle, and increased ethylene and salicylic acid (SA) levels. Thus, we proposed that the N sources reprogrammed C and N metabolism, attenuating the competition of N assimilation for C, and promoting the synthesis and accumulation of andrographolide through plant hormone signaling. To obtain a higher production of andrographolide in A. paniculata, AN fertilizer is recommended in its N management.

Improved Cordycepin Production by Cordyceps Militaris Using Corn Steep Liquor Hydrolysate as an Alternative Protein Nitrogen Source.

Cordycepin production in the submerged culture of Cordyceps militaris was demonstrated using hydrolyzed corn processing protein by-products, known as corn steep liquor hydrolysate (CSLH), as an alternative nitrogen source. The growth, metabolism, and cordycepin production of Cordyceps militaris were evaluated under various concentrations of CSLH induction. The results demonstrated that CSLH addition had positive effects on the growth and cordycepin production with various C. militaris strains. The optimum strain, C. militaris GDMCC5.270, was found to effectively utilize CSLH to promote mycelium growth and cordycepin production. Low concentrations of CSLH (1.5 g/L) in the fermentation broth resulted in 343.03 ± 15.94 mg/L cordycepin production, which was 4.83 times higher than that of the group without CSLH. This also enhanced the metabolism of sugar, amino acids, and nucleotides, leading to improved cordycepin biosynthesis. The increase in key amino acids, such as glutamic acid, alanine, and aspartic acid, in the corn steep liquor hydrolysate significantly enhanced cordycepin yield. The corn steep liquor hydrolysate was confirmed to be a cost-effective accelerator for mycelium growth and cordycepin accumulation in C. militaris, replacing partial peptone as a cheap nitrogen source. It serves as a suitable alternative for efficient cordycepin production at a low cost.

Levan Production by Suhomyces kilbournensis Using Sugarcane Molasses as a Carbon Source in Submerged Fermentation.

The valorization of byproducts from the sugarcane industry represents a potential alternative method with a low energy cost for the production of metabolites that are of commercial and industrial interest. The production of exopolysaccharides (EPSs) was carried out using the yeast Suhomyces kilbournensis isolated from agro-industrial sugarcane, and the products and byproducts of this agro-industrial sugarcane were used as carbon sources for their recovery. The effect of pH, temperature, and carbon and nitrogen sources and their concentration in EPS production by submerged fermentation (SmF) was studied in 170 mL glass containers of uniform geometry at 30 °C with an initial pH of 6.5. The resulting EPSs were characterized with Fourier-transform infrared spectroscopy (FT-IR). The results showed that the highest EPS production yields were 4.26 and 44.33 g/L after 6 h of fermentation using sucrose and molasses as carbon sources, respectively. Finally, an FT-IR analysis of the EPSs produced by S. kilbournensis corresponded to levan, corroborating its origin. It is important to mention that this is the first work that reports the production of levan using this yeast. This is relevant because, currently, most studies are focused on the use of recombinant and genetically modified microorganisms; in this scenario, Suhomyces kilbournensis is a native yeast isolated from the sugar production process, giving it a great advantage in the incorporation of carbon sources into their metabolic processes in order to produce levan sucrose, which uses fructose to polymerize levan.

The Nitrogen Content in the Fruiting Body and Mycelium of Pleurotus Ostreatus and Its Utilization as a Medium Component in Thraustochytrid Fermentation.

Following the idea of a circular bioeconomy, the use of side streams as substitutes for cultivation media (components) in bioprocesses would mean an enormous economic and ecological advantage. Costly compounds in conventional media for the production of the triterpene squalene in thraustochytrids are the main carbon source and complex nitrogen sources. Among other side streams examined, extracts from the spent mycelium of the basidiomycete Pleurotus ostreatus were best-suited to acting as alternative nitrogen sources in cultivation media for thraustochytrids. The total nitrogen (3.76 ± 0.01 and 4.24 ± 0.04%, respectively) and protein (16.47 ± 0.06 and 18.57 ± 0.18%, respectively) contents of the fruiting body and mycelium were determined. The fungal cells were hydrolyzed and extracted to generate accessible nitrogen sources. Under preferred conditions, the extracts from the fruiting body and mycelium contained 73.63 ± 1.19 and 89.93 ± 7.54 mM of free amino groups, respectively. Cultivations of Schizochytrium sp. S31 on a medium using a mycelium extract as a complex nitrogen source showed decelerated growth but a similar squalene yield (123.79 ± 14.11 mg/L after 216 h) compared to a conventional medium (111.29 ± 19.96 mg/L, although improvable by additional complex nitrogen source).

Enhancing surfactin production in Bacillus subtilis: Insights from proteomic analysis of nitrate-induced overproduction and strategies for combinatorial metabolic engineering.

Surfactin biosynthesis in Bacillus subtilis is intricately regulated by environmental conditions. In the present study, addition of nitrate, a nitrogen source, increased the production of surfactin in B. subtilis ATCC 21332, whereas its absence resulted in minimal or no surfactin production. Proteomics revealed the mechanism underlying nitrate-induced surfactin overproduction, identifying three key differential proteins (preprotein translocase subunit SecA, signal recognition particle receptor FtsY, and cell division adenosine triphosphate-binding protein FtsE) relevant to surfactin transport and regulation. Combinatorial metabolic engineering strategies (enhanced nitrate reduction, fatty acid hydroxylation, rational transporter engineering, and feeding) led to a 41.4-fold increase in surfactin production compared with the initial production in the wild-type strain. This study provides insights into the molecular mechanism of nitrate-induced surfactin overproduction and strategies to enhance the performance of surfactin-producing strains.

Enhanced sporulation of B. licheniformis BF-002 through automatic co-feeding of carbon and nitrogen sources.

Bacillus licheniformis formulations are effective for environmental remediation, gut microbiota modulation, and soil improvement. An adequate spore quantity is crucial for the activity of B. licheniformis formulations. This study investigated the synergistic effects of carbon/nitrogen source consumption and concentration on B. licheniformis BF-002 cultivation, with the aim of developing an automatic co-feeding strategy to enhance spore production. Initial glucose (10 g/L) and amino nitrogen (1.5 g/L) concentrations promote cell growth, followed by reduced glucose (2.0 g/L) and amino nitrogen (0.5 g/L) concentrations for sustained spore generation. The spore quantity reached 2.59 × 1010 CFU/mL. An automatic co-feeding strategy was developed and implemented in 5 and 50 L cultivations, resulting in spore quantities of 2.35 × 1010 and 2.86 × 1010 CFU/mL, respectively, improving by 6.81% and 30.00% compared to that with a fixed glucose concentration (10.0 g/L). The culture broth obtained at both the 5 and 50 L scales was spray-dried, resulting in bacterial powder with cell viability rates of 85.94% and 82.68%, respectively. Even after exposure to harsh conditions involving high temperature and humidity, cell viability remained at 72.80% and 69.89%, respectively. Employing the automatic co-feeding strategy increased the transcription levels of the spore formation-related genes spo0A, spoIIGA, bofA, and spoIV by 7.42%, 8.46%, 8.87%, and 9.79%, respectively. The proposed strategy effectively promoted Bacillus growth and spore formation, thereby enhancing the quality of B. licheniformis formulations.

Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae.

Nitrogen source assimilation is important for the biological functions of fungi, and its pathway has been deeply studied. Aspergillus oryzae mutants defective in nitrogen source assimilation are known to grow poorly on Czapek-Dox (CD) medium. In this study, we found an industrial strain of A. oryzae that grew very poorly on a CD medium containing sodium nitrate as a nitrogen source. We used media with various nitrogen components to examine the steps affecting the nitrogen source assimilation pathway of this strain. The strain grew well on the CD medium supplied with nitrite salt or ammonium salt, suggesting that the strain was defective in nitrate assimilation step. To ascertain the gene causing the defect of nitrate assimilation, a gene expression vector harboring either niaD or crnA of A. oryzae RIB40 was introduced into the industrial strain. The industrial strain containing the crnA vector recovered its growth. This is the first report that a mutation of crnA causes poor growth on CD medium in an industrial strain of A. oryzae, and crnA can be used as a transformation marker for crnA deficient strains.

Prospects of formamide as nitrogen source in biotechnological production processes.

This review presents an analysis of formamide, focussing on its occurrence in nature, its functional roles, and its promising applications in the context of the bioeconomy. We discuss the utilization of formamide as an innovative nitrogen source achieved through metabolic engineering. These approaches underscore formamide's potential in supporting growth and production in biotechnological processes. Furthermore, our review illuminates formamide's role as a nitrogen source capable of safeguarding cultivation systems against contamination in non-sterile conditions. This attribute adds an extra layer of practicality to its application, rendering it an attractive candidate for sustainable and resilient industrial practices. Additionally, the article unveils the versatility of formamide as a potential carbon source that could be combined with formate or CO2 assimilation pathways. However, its attributes, i.e., enriched nitrogen content and comparatively limited energy content, led to conclude that formamide is more suitable as a co-substrate and that its use as a sole source of carbon for biomass and bio-production is limited. Through our exploration of formamide's properties and its applications, this review underscores the significance of formamide as valuable resource for a large spectrum of industrial applications. KEY POINTS: • Formidases enable access to formamide as source of nitrogen, carbon, and energy • The formamide/formamidase system supports non-sterile fermentation • The nitrogen source formamide supports production of nitrogenous compounds.

Optimal Laboratory Cultivation Conditions of Limnospira maxima for Large-Scale Production.

Cultivating Limnospira maxima, renowned for its abundant proteins and valuable pigments, faces substantial challenges rooted in the limited understanding of its optimal growth parameters, associated high costs, and constraints in the procurement of traditional nitrogen sources, particularly NaNO3. To overcome these challenges, we conducted a comprehensive 4 × 3 factorial design study. Factors considered included white, red, blue, and yellow light spectra, along with nitrogen sources NaNO3 and KNO3, as well as a nitrogen-free control, for large-scale implementation. Optimal growth, measured by Optical Density, occurred with white and yellow light combined with KNO3 as the nitrogen source. These conditions also increased dry weight and Chl-a content. Cultures with nitrogen deprivation exhibited high values for these variables, attributed to carbon accumulation in response to nitrogen scarcity. Phycocyanin, a crucial pigment for nutrition and industry, reached its highest levels in cultures exposed to white light and supplemented with KNO3, with an impressive content of 384.11 g kg-1 of dry weight. These results highlight the efficacy and cost-efficiency of using a combination of white light and KNO3 for large-scale L. maxima cultivation. This strategy offers promising opportunities to address global food security challenges and enhance the production of industrially relevant pigments.

Comprehensive Exploration of the Growth and Lipid Synthesis Phases of T. oleaginosus Cultures Implementing Design of Experiments and Response Surface Methodology.

Trichosporon oleaginosus is an unconventional oleaginous yeast distinguished by its remarkable capacity to accumulate lipids in excess of 70% of its dry weight, particularly when cultivated in nitrogen-restricted conditions with ample carbon sources. A pivotal question that arises pertains to the nutrient dynamics in the culture medium, which give rise to both the excessive lipid content and corresponding lipid concentration. While previous research has predominantly focused on evaluating the impact of the initial carbon-to-nitrogen (C/N) ratio on lipid production, the precise critical thresholds of glucose and ammonium sulfate ((NH4)2SO4) at which growth and intracellular lipid production are either stimulated or impeded remain inadequately defined. This study employs an experimental design and response surface methodology to investigate the complex mechanism of lipid accumulation and its interaction with cellular growth. Application of the aforementioned methodologies resulted in the production of 10.6 g/L of microbial oil in batch cultures under conditions that correspond to a C/N ratio of 76. However, the primary objective is to generate knowledge to facilitate the development of efficient fed-batch cultivation strategies that optimize lipid production exclusively employing inorganic nitrogen sources by finely adjusting carbon and nitrogen levels. The intricate interaction between these levels is comprehensively addressed in the present study, while it is additionally revealed that as glucose levels rise within a non-inhibitory range, lipid-free biomass production decreases while lipid accumulation simultaneously increases. These findings set the stage for further exploration and the potential development of two-stage cultivation approaches, aiming to fully decouple growth and lipid production. This advancement holds the promise of bringing microbial oil production closer to commercial viability.

Potential of algal oil production from secondary treated sewage: a study using Chlorella vulgaris and synthetic wastewater.

To assess the potential of using the secondary treated wastewater for the production of algal biofuel, batch experiments were carried out in photobioreactors using indigenous Chlorella vulgaris isolated from the natural freshwater body. Secondary treated wastewater with partial nitrification was simulated using various proportions of NO3-N and NH4-N while keeping the total nitrogen the same. Experiments with similar concentrations of nitrate without the NH4-N were used for comparison. In the presence of only NO3-N in the concentration range of 9-37 mg/L, the growth and fatty acid methyl ester (FAME) production was similar to the literature reports. When NH4-N was present along with NO3-N, the biomass growth was adversely affected, indicating an impact on the metabolic activity. For the same initial concentrations of nitrate in the culture, the maximum biomass concentration was reduced by 50-60% in the presence of NH4-N. The FAME profile changed significantly and a new FAME was identified, suggesting an impact on the lipid synthesis pathway. Comparison and analysis with the help of existing literature indicated that the adverse effect due to NH4-N was a function of pH. The growth, biomass yield, and FAME production were unaffected by a wide range of phosphorus concentrations. Maximum fatty acid methyl ester (FAME) suitable for biodiesel production (fatty acid carbon chain length C16 to C18) was 381.01 mg/L (224.58 mg/g of dry biomass), produced at NO3-N concentration of 18.5 mg/L and initial nitrogen loading per unit biomass of 0.37 g NO3-N/g of dry biomass.

CAR1 as a new selective marker for genetic engineering of wine yeasts.

A common problem in engineering industrial yeasts, and wine yeasts in particular, is the lack or scarcity of selective markers for introducing desired genetic changes. Almost all such markers, which are usually auxotrophic mutations, would reduce the growth characteristics of yeast strains. However, a potentially useful marker could be the CAR1 gene encoding arginase, the deletion of which reduces the accumulation of the carcinogen ethyl carbamate in wine, making such a deletion beneficial for wine production and maintainable in wine yeast strains. Here we demonstrate the use of the CAR1 gene as a selective marker. First, we observe that complete deletion of CAR1 in a triploid wine strain of Saccharomyces cerevisiae causes strong growth inhibition on a medium containing arginine as the only nitrogen source. Then, we show that strains with CAR1 deletion can be reliably transformed using CAR1 as a plasmid marker. Thus, the CAR1 gene can be used as a convenient selective marker in genetic engineering of wine yeasts, in particular using CRISPR/Cas9 technology.

Optimization of Heterotrophic Culture Conditions for the Microalgae Euglena gracilis to Produce Proteins.

Euglena gracilis is one of the few permitted edible microalgae. Considering consumer acceptance, E. gracilis grown heterotrophically with yellow appearances have wider food industrial applications such as producing meat analogs than green cells. However, there is much room to improve the protein content of heterotrophic culture cells. In this study, the effects of nitrogen sources, temperature, initial pH, and C/N ratios on the protein production of E. gracilis were evaluated under heterotrophic cultivation. These results indicated that ammonium sulfate was the optimal nitrogen source for protein production. The protein content of E. gracilis cultured by ammonium sulfate increased by 113% and 44.7% compared with that cultured by yeast extract and monosodium glutamate, respectively. The manipulation of the low C/N ratio further improved E. gracilis protein content to 66.10% (w/w), which was 1.6-fold of that in the C/N = 25 group. Additionally, amino acid analysis revealed that the nitrogen-to-protein conversion factor (NTP) could be affected by nitrogen sources. A superior essential amino acid index (EAAI) of 1.62 and a balanced amino acid profile further confirmed the high nutritional value of E. gracilis protein fed by ammonium sulfate. This study highlighted the vast potency of heterotrophic cultured E. gracilis as an alternative dietary protein source.

Mathematical Estimation of Endogenous Proline as a Bioindicator to Regulate the Stress of Trivalent Chromium on Rice Plants Grown in Different Nitrogenous Conditions.

The accumulation of proline impacts the defense mechanisms of plants against the harmful effects of adverse environmental conditions; however, its concentration in plants is associated with the metabolism of N. Therefore, the effects of exogenous organic [glutamate (Glu)/arginine (Arg)] and inorganic [nitrate (NO3-)/ammonium (NH4+)] N on the accumulation of proline (Pro) in rice plants under trivalent chromium [Cr(III)] stress were studied through using the mass balance matrix model (MBMM). Application of 'NH4+' showed the largest contribution to the Pro content in rice shoots under different concentrations of Cr(III), followed by 'NO3-', 'Arg', and 'Glu' applications. On the other hand, 'Arg' application displayed the largest contribution to the Pro content in roots under Cr(III) stress, followed by 'NH4+', 'Glu', and 'NO3-' applications. The combined application of 'NH4++Arg' showed the greatest contribution to the Pro content in both roots and shoots of Cr(III)-treated rice seedlings, while the application of 'NO3-+Glu' showed the least contribution to the Pro content in rice seedlings. The current study indicated that the endogenous level of Pro in rice seedlings is quite sensitive to Cr(III) stress under different N sources, and the mathematical modeling showed a reliable result while estimating the relationship between Pro content and N source application.

Sources and transformation of nitrogen in shallow aquifers with progressive water table recovery using geochemical and isotopic approaches.

Groundwater level recovery has a significant effect on the sources and transformation of nitrogen in groundwater, but there are still few studies on the influences of the water table on the sources and transformation of nitrogen in groundwater using field data. In this study, the changes in groundwater level, geochemical composition, and isotopic signatures of NO3- and NH4+ during a period of groundwater level recovery at a pilot site were analyzed in detail. The water table underwent progressive recovery of almost 1.6 m in 16 months. At a depth of 5.5 m below the surface, both low NH4+ and high NO3- appeared in the groundwater, whereas below that depth, both high NH4+ and low NO3- simultaneously appeared in the groundwater. The main sources of NO3- were manure and septic waste, and NH4+ fertilizers. The main sources of NH4+ were mineral fertilizers. The main transformation process were nitrification and denitrification at a depth of 5.5 m below the surface; below that depth, the main transformation processes were denitrification and dissimilatory nitrate reduction to ammonium (DNRA). This study provides a theoretical understanding of the relationship between the changing water table and nitrogen in groundwater level recovery areas.

Carbon and nitrogen sources consumption by ale and lager yeast strains: a comparative study during fermentation.

The rapid and efficient consumption of carbon and nitrogen sources by brewer's yeast is critical for the fermentation process in the brewing industry. The comparison of the growth characterizations of typical ale and lager yeast, as well as their consumption preference to carbon and nitrogen sources were investigated in this study. Results showed that the ale strain grew faster and had a more extended stationary phase than the lager strain. However, the lager strain was more tolerant to the stressful environment in the later stage of fermentation. Meanwhile, the ale and lager yeast strains possessed varying preferences for metabolizing the specific fermentable sugar or free amino acid involved in the wort medium. The lager strain had a strong capacity to synthesize the extracellular invertase required for hydrolyzing sucrose as well as a strong capability to metabolize glucose and fructose. Furthermore, the lager strain had an advantage in consuming Lys, Arg, Val, and Phe, whereas the ale strain had a higher assimilation rate in consuming Tyr. These findings provide valuable insights into selecting the appropriate brewer's yeast strain based on the wort components for the industrial fermentation process. KEY POINTS: • The lager strain is more tolerant to the stressful environment. • The lager strain has the great capability to synthesize the extracellular invertase. • The assimilation efficiency of free amino acid varies between ale and lager.

Supplementing the Nuclear-Encoded PSII Subunit D1 Induces Dramatic Metabolic Reprogramming in Flag Leaves during Grain Filling in Rice.

Our previous study has demonstrated that the nuclear-origin supplementation of the PSII core subunit D1 protein stimulates growth and increases grain yields in transgenic rice plants by enhancing photosynthetic efficiency. In this study, the underlying mechanisms have been explored regarding how the enhanced photosynthetic capacity affects metabolic activities in the transgenic plants of rice harboring the integrated transgene RbcSPTP-OspsbA cDNA, cloned from rice, under control of the AtHsfA2 promoter and N-terminal fused with the plastid-transit peptide sequence (PTP) cloned from the AtRbcS. Here, a comparative metabolomic analysis was performed using LC-MS in flag leaves of the transgenic rice plants during the grain-filling stage. Critically, the dramatic reduction in the quantities of nucleotides and certain free amino acids was detected, suggesting that the increased photosynthetic assimilation and grain yield in the transgenic plants correlates with the reduced contents of free nucleotides and the amino acids such as glutamine and glutamic acid, which are cellular nitrogen sources. These results suggest that enhanced photosynthesis needs consuming more free nucleotides and nitrogen sources to support the increase in biomass and yields, as exhibited in transgenic rice plants. Unexpectedly, dramatic changes were measured in the contents of flavonoids in the flag leaves, suggesting that a tight and coordinated relationship exists between increasing photosynthetic assimilation and flavonoid biosynthesis. Consistent with the enhanced photosynthetic efficiency, the substantial increase was measured in the content of starch, which is the primary product of the Calvin-Benson cycle, in the transgenic rice plants under field growth conditions.

Inhibition of Aspergillus carbonarius growth and Ochratoxin A production using lactic acid bacteria cultivated in an optimized medium.

The Gram-positive bacteria lactic acid bacteria (LAB) are used in the food industry but are also known for inhibiting certain food spoilage microorganisms, especially fungi. Sources of nitrogen (N) for culture media are generally organic and expensive. Many attempts have been made to formulate economical culture media with alternative N sources obtained from agricultural and industrial byproducts. This study describes the design and optimization of an inexpensive culture medium for Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) MZ809351 strain B31. The culture medium was optimized using statistical experimental designs to identify the factors with the most significant effects on biomass concentration to reduce the overall cost, aiming to obtain a biomass concentration similar to that obtained with the reference LAB culture medium (de Man, Rogosa and Sharpe; MRS). Sodium acetate and magnesium sulfate were the most significant factors (p < 0.005), and their contents were reduced by 22 % and 40 %, respectively, without affecting biomass concentration. Malt germ extract (MGE) was used as an alternative nitrogen source to replace meat extract (ME) and proteose peptone (PP). Through these experiments, the composition of a culture medium that is less expensive than MRS broth was defined, which produced a biomass concentration (3.8 g/L) similar to that obtained with MRS medium. The inhibitory effects of two LAB strains isolated from the Ivory Coast and Mexico on the growth and production of ochratoxin A (OTA) in an ochratoxigenic fungus was tested. The minimum cellular concentration of the LAB to prevent the development of Aspergillus carbonarius Ac 089 and the production of OTA was determined in a model assay in Petri dishes. The conditions to inhibit the germination of A. carbonarius Ac 089 and the production of OTA were found. Using the optimized medium and a ratio of 2 × 104 LAB/spore (1 × 108 CFU/mL) strain B7 (L. plantarum MZ809351) and 2 × 103 LAB/spore (1 × 107 CFU/mL) strain B31 (L. plantarum MN922335) completely inhibited the growth of the fungus. A ratio of 2 × 105 LAB/spore (1 × 109 CFU/mL) was required to inhibit OTA production with strains B7 and B31. This study indicates the potential of cultivating LAB in an optimized and inexpensive culture medium for use as a biological control agent against ochratoxigenic fungi in food.

A Chemically Defined Medium That Supports Mycotoxin Production by Stachybotrys chartarum Enabled Analysis of the Impact of Nitrogen and Carbon Sources on the Biosynthesis of Macrocyclic Trichothecenes and Stachybotrylactam.

Stachybotrys chartarum (Hypocreales, Ascomycota) is a toxigenic fungus that is frequently isolated from water-damaged buildings or improperly stored feed. The secondary metabolites formed by this mold have been associated with health problems in humans and animals. Several authors have studied the influence of environmental conditions on the production of mycotoxins, but these studies focused on undefined or complex substrates, such as building materials and media that impeded investigations of the influence of specific nutrients. In this study, a chemically defined cultivation medium was used to investigate the impact of several nitrogen and carbon sources on growth of S. chartarum and its production of macrocyclic trichothecenes (MTs) and stachybotrylactam (STLAC). Increasing concentrations of sodium nitrate were found to positively affect mycelial growth, the level of sporulation, and MT production, while ammonium nitrate and ammonium chloride had an inhibitory effect. Potato starch was the superior and most reliable carbon source tested. Additionally, we observed that the level of sporulation was correlated with the production of MTs but not with that of STLAC. In this study, we provide a chemically well-defined cultivation medium suitable for standardized in vitro testing of the capacity of S. chartarum isolates to produce macrocyclic trichothecenes. IMPORTANCE Macrocyclic trichothecenes (MTs) are highly toxic secondary metabolites that are produced by certain Stachybotrys chartarum strains, which consequently pose a risk for animals and humans. To identify hazardous, toxin-producing strains by analytical means, it is important to grow them under conditions that support MT production. Nutrients determine growth and development and thus the synthesis of secondary metabolites. Complex rich media are commonly used for diagnostics, but batch differences of supplements pose a risk for inconsistent data. We have established a chemically defined medium for S. chartarum and used it to analyze the impact of nitrogen and carbon sources. A key finding is that nitrate stimulates MT production, whereas ammonium suppresses it. Defining nutrients that support MT production will enable a more reliable identification of hazardous S. chartarum isolates. The new medium will also be instrumental in analyzing the biosynthetic pathways and regulatory mechanisms that control mycotoxin production in S. chartarum.

Nitrogen sources alter ligninase and cellulase activities of thermophilic fungi isolated from compost and vermicompost.

Fungi harboring lignocellulolytic activity accelerate the composting process of agricultural wastes; however, using thermophilic fungal isolates for this process has been paid little attention. Moreover, exogenous nitrogen sources may differently affect fungal lignocellulolytic activity. A total of 250 thermophilic fungi were isolated from local compost and vermicompost samples. First, the isolates were qualitative assayed for ligninase and cellulase activities using Congo red (CR) and carboxymethyl cellulose (CMC) as substrates, respectively. Then, twenty superior isolates harboring higher ligninase and cellulase activities were selected and quantitatively assayed for both enzymes in basic mineral (BM) liquid medium supplemented with the relevant substrates and nitrogen sources including (NH4)2SO4 (AS), NH4NO3 (AN), urea (U), AS + U (1:1), or AN + U (1:1) with final nitrogen concentration of 0.3 g/L. The highest ligninase activities of 99.94, 89.82, 95.42, 96.25, and 98.34% of CR decolorization were recorded in isolates VC85, VC94, VC85, C145, and VC85 in the presence of AS, U, AS + U, AN, and AN + U, respectively. Mean ligninase activity of 63.75% in superior isolates was achieved in the presence of AS and ranked the highest among other N compounds. The isolates C200 and C184 exhibited the highest cellulolytic activity in the presence of AS and AN + U by 8.8 and 6.5 U/ml, respectively. Mean cellulase activity of 3.90 U/mL was achieved in AN + U and ranked the highest among other N compounds. Molecular identification of twenty superior isolates confirmed that all of them are belonging to Aspergillus fumigatus group. Focusing on the highest ligninase activity of the isolate VC85 in the presence of AS, the combination can be recommended as a potential bio-accelerator for compost production.