Nitrogen Source Research Articles

Production of recombinant human insulin from a promising Pseudomonas fluorescens cell factory and its kinetic modeling

Insulin intake is recommended for diabetics in addition to a proper diet and lifestyle to maintain adequate blood glucose level. Currently, there is a need for an alternative expression system for insulin production as the current expression systems may not meet the growing demand due to various constraints. Here, we demonstrate the synthesis of human insulin in an unconventional expression system based on Pseudomonas fluorescens, a BSL 1 bacterium. Human insulin was produced in the form of proinsulin fused with fusion protein. Then, the proinsulin fusion protein was purified using Ni-NTA chromatography and converted into human insulin. The physicochemical parameters for producing proinsulin fusion protein are optimized. Glucose and ammonium chloride are determined to be suitable carbon and nitrogen sources, respectively. The validity of insulin and proinsulin fusion protein is assessed using western blot and quantified using ELISA techniques. Up to 145.35 mg/l of the proinsulin fusion protein is achieved at the shake flask level. Further, MALDI-TOF and RP-HPLC analysis of the purified human insulin were observed to be close to the theoretical value and insulin standard, respectively. The expression of the recombinant fusion protein was found to be 214.7 mg/l in a batch bioreactor, a ∼48% enhancement over the shake flask level. Further, kinetic modeling was performed to understand the system regarding growth, substrate utilization and product formation, and to estimate the various kinetic parameters. This study establishes the potential of the P. fluorescens expression system for producing human insulin.

إنتاج الليبازالبارد – النشط بواسطة فيوزاريوم سولاني

The current study aimed to the production and partial purification of a cold-active lipase by some fungi isolated from the olive oil processing wastes in Al-Gabal Al-Gharby, Libya. 31 fungal species from 12 genera were isolated. F.solani was the most prevalent comprising 94% of total Fusarium and 28.7% of total fungi, 102 fungal isolates were tested for their lipolytic activity on lipase production agar medium at 10 and 20°C. The most active isolates were Alternaria (2 isolates), Fusarium, and Penicillium (1isolate for each one). Molecular identification of the most active four isolates was carried out by their sequencing (ITS). The four powerful fungal strains' production of cold-active lipase was maximized by optimizing some nutritional and environmental factors. F. solani AUMC 16063 was able to produce the maximum amount of lipase activity (46.66U/mL/min) with specific activity (202.8U/mg), utilizing ammonium sulphate as a nitrogen source after 8 days of incubation at pH 3.0 and 15°C. However, at same condition after 6 days when yeast extract was employed as a nitrogen source, the generated cold-active lipase displayed the highest specific activity of (1550U/mg) and lipase activity (36.74U/ml/min). This is the first study in which the production, partial purification, maximized and characterization of a cold-active lipase enzyme by Fusarium solani.

Comparison of Amino Acid Digestibility between Commercial Crossbred Pigs and Mini-Jeju Island Native Pigs.

The objectives of this study were to determine the apparent ileal digestibility and standardized ileal digestibility (SID) of crude protein (CP) and amino acids (AA) in feed ingredients, compare the ileal digestibility of CP and AA between commercial crossbred pigs and mini-Jeju Island native pigs (JINP), and develop models for estimating SID of CP and AA for commercial pigs using mini-JINP data. The study involved five crossbred commercial pigs (31.5 ± 1.6 kg of body weight and 11 weeks of age; Landrace × Yorkshire) and five mini-JINP (31.0 ± 3.2 kg body weight and 20 weeks of age). The pigs were surgically equipped with a T-cannula at the end of ileum. Each pig breed was assigned to 5 dietary treatments in a 5 × 10 incomplete Latin square design with 10 periods. Four experimental diets were formulated to contain each of soybean meal, corn gluten feed, copra meal, and sesame expellers as the sole source of nitrogen. A nitrogen-free diet was also prepared to determine basal endogenous losses of CP and AA. No interaction between breed and feed ingredient was observed for the digestibility of CP and all indispensable AA. The SID of CP and all indispensable AA, except Arg, His, and Lys, did not differ between the two breeds of pigs. Prediction equations were developed for SID of CP and AA of commercial pigs using the SID values of mini-JINP: SID of CP (%) = (1.02 × SID of CP in mini-JINP) - 5.20 with r2 = 0.97 and p < 0.05; SID of Lys (%) = (1.12 × SID of Lys in mini-JINP) - 9.10 with r2 = 0.98 and p < 0.05; and SID of Met (%) = (1.08 × SID of Met in mini-JINP) - 4.27 with r2 = 0.96 and p < 0.05. The digestibility for most AA in feedstuffs for commercial pigs can be estimated using data from mini-JINP.

РОСТСТИМУЛИРУЮЩАЯ АКТИВНОСТЬ БАКТЕРИЙ ENTEROBACTER LUDWIGII BLK ПРИ ОПТИМИЗАЦИИ СРЕДЫ КУЛЬТИВИРОВАНИЯ

A method for optimizing the cultivation of Enterobacter ludwigii BLK and the treatment of wheat plants using a preparation based on this bacterium is proposed. By reducing the concentration of peptone and partially replacing it with an alternative nitrogen source – yeast extract – in the culture medium, the cost of producing the drug was reduced without reducing its effectiveness. It was found that the preparation, produced in a modified environment, activated root growth and promoted the accumulation of chlorophyll in bread wheat leaves. The stimulating effect was more pronounced with a combination of pre-sowing and post-emergence treatment.

Optimized medium conditions maximize colony regeneration from a single cell of Botryococcus braunii NIES836

Botryococcus braunii is a colonial alga recognized for its slow growth but high hydrocarbon accumulation. Although using genetic engineering to increase the growth rate and hydrocarbon yield of B. braunii is desirable, the presence of an extracellular matrix (ECM) significantly hinders the emergence of a homogeneous colony from a single DNA-transformed cell. Previously, we developed a method to isolate single cells without ECM from colonies. However, following the isolation of single cells, several months are required to regenerate colonies with a sufficient cell mass for subsequent analysis. To shorten the colony regeneration period, we investigated basal media and medium components, along with growth-promoting additives, in a series of single-factor experiments and optimized the concentrations of the medium constituents via response surface methodology (RSM). The results of the single-factor experiments revealed that the nitrogen source (a mixture of NaNO3 and NH4NO3), 1-naphthylacetic acid (NAA) and Fe(III)-citrate significantly increased the growth of B. braunii single cells into colonies. The optimal medium composition identified by RSM included 151.6 mg/L nitrogen source, 2.419 mg/L NAA and 15.3 mg/L Fe(III)-citrate. Verification experiments showed that the optimized medium resulted in a 1.75-fold increase in colony size compared with that of colonies grown in nonoptimized AF6 medium. This is the first report of the optimal medium composition for the regeneration of B. braunii colonies from single cells.

Stimulation of citric acid production by heat shock promoting &lt;i&gt;Aspergillus niger&lt;/i&gt; using nitrogen enriched &lt;i&gt;Dioscorea bulbifera&lt;/i&gt;

High cost of substrate and how to stimulate citric acid are among the factors that mitigate citric acid production. Exploiting a cheap substrate for citric acid production will be a viable alternative in order to reduce the cost of citric acid production. Therefore, the aim of this research was to evaluate the potential of wild Dioscorea bulbifera tubers for citric acid production. Experimental procedures were designed to determine the effect of different concentrations of wild Dioscorea bulbifera, different nitrogen sources and effect of different concentrations of nitrogen sources on citric acid production. The data obtained were analyzed using one- way analysis of variance (ANOVA). The results of the experiment showed that wild Dioscorea bulbifera tuber was able to produce citric acid. The concentration of acid produced increased from 5 % to 20 % citric acid up to 8.5 ±1.0 g/lafter 96 hours of fermentation. Among the different nitrogen sources used to supplement wild Dioscorea bulbifera, the medium with ammonium nitrate produced the highest citric acid concentration of 10.5 ±1.5 g/l after 96 hours of fermentation. Meanwhile, when the concentration of the amonium nitrate were varied, 0.4 % was the optimum for the maximum citric acid concentration of 14.8 ±2.0 g/l after 96 hours of fermentation (P&lt; 0.05). In conclusion, Dioscorea bulbifera tubers is suitable for citric acid production and supplementation of the medium with ammonium nitrate as a nitrogen source had a positive effect on the yield.

Inclusion of cool‐ and warm‐season species in tall fescue swards for increased productivity

AbstractPasture systems that include cool‐ and warm‐season species have the potential to enhance tall fescue (TF) [Schedonorus arundinaceus (Schreb.) Dumort.] forage systems beyond their typical season. The objectives of this study were to incorporate crabgrass (Digitaria ciliaris Retz.) into TF swards and compare the production of monoculture TF to a binary mixture of crabgrass and TF as well as its effect with different sources of nitrogen (such as red clover [Trifolium pratense L.] or sunn hemp [Crotalaria juncea L.]). The experiment was conducted in Crossville, TN, in 2020 and 2021. The treatments were as follows: (1) TF + 0 N (T), (2) TF + ammonium nitrate (TA), (3) TF + red clover (TR), (4) TF + sunn hemp (TS), (5) TF + crabgrass + 0 N (TFC), (6) TF + crabgrass + ammonium nitrate (TCA), (7) TF + crabgrass + red clover (TCR), and (8) TF + crabgrass + sunn hemp (TCS). The plots containing red clover indicated a greater herbage mass (HM) and crude protein (CP) along with lesser neutral detergent fiber compared to the other treatments. Sunn hemp performed best during the mid‐summer, contributing to the increase of HM. Tall fescue swards that were mixed with crabgrass and a source of N (legumes or N fertilizer) had greater HM during the warm season, having the potential to decrease the stationarity of production. Thus, including crabgrass, red clover, and sunn hemp (grass and legumes, respectively) in TF pasture can be a great strategy to increase HM and improve nutritive value.

Seawater spray as a significant nitrogen source across coastal dune vegetation gradients

BackgroundNitrogen significantly influences plant performance and vegetation development in nutrient-poor ecosystems like coastal dunes. While various sources contribute nitrogen, including N2 fixation and marine inputs, the significance of seawater spray remains understudied. In this study, we aimed to assess the relevance of seawater spray as a source of nitrogen input and its potential role in plant community composition in dune ecosystems. MethodsThe δ15N, δ13C, N, and C content of leaves from the most abundant 21 species were measured in 6 positions across a beach inland gradient in a Mediterranean dune system in SW Spain. Soil samples at different depths were collected in each position and N, C, P, K, NH4+, NO3=, and organic matter were measured. Salt spray accumulation was determined on Achillea maritima leaves across the gradient. ResultsLeaf nitrogen content did not exhibit a beach-inland gradient, but δ15N decreased with distance from the sea. Species displayed three distinct N uptake strategies along the gradient: species from Upper Beach and Foredune communities showed high δ15N values, suggesting a marine origin; species distributed across the gradient exhibited decreasing δ15N patterns from the Upper Beach to the Inland, indicative of seawater spray influence; species farthest from the sea relied on non-marine nitrogen sources. ConclusionsThese results indicate the importance of seawater nitrogen income for the dune system vegetation and evidence that dune plant species exhibit varied N uptake strategies influenced by their position across the beach-inland gradient.

Hydroxylamine‐O‐Sulfonic Acid (HOSA): A Recent Synthetic Overview

AbstractHydroxylamine‐O‐sulfonic acid has been used as a versatile nitrogen source in synthetic organic chemistry. It has dual function and utilized both as a nucleophile and an electrophile during different synthetic transformations. This reagent is stable and easy to handle in both aqueous and organic solvents. It can be used with different functional groups under mild reaction conditions, making its widely applicable for constructing complex molecules. It has been utilized for various applications such as agrochemicals, pharmaceuticals, and polymer industries in the formation of S−N, C−N, O−N, and N−N bonds, as well as for efficient cyclization reactions under metal‐free conditions with excellent stereo– and regioselectivity. The present review provides a brief overview of all the advancements made in the past two decades using hydroxylamine‐O‐sulfonic acid as a nitrogen source.

Transcriptomic analyses of bacterial growth on fungal necromass reveal different microbial community niches during degradation.

Bacteria are major drivers of organic matter decomposition and play crucial roles in global nutrient cycling. Although the degradation of dead fungal biomass (necromass) is increasingly recognized as an important contributor to soil carbon (C) and nitrogen (N) cycling, the genes and metabolic pathways involved in necromass degradation are less characterized. In particular, how bacteria degrade necromass containing different quantities of melanin, which largely control rates of necromass decomposition in situ, is largely unknown. To address this gap, we conducted a multi-timepoint transcriptomic analysis using three Gram-negative, bacterial species grown on low or high melanin necromass of Hyaloscypha bicolor. The bacterial species, Cellvibrio japonicus, Chitinophaga pinensis, and Serratia marcescens, belong to genera known to degrade necromass in situ. We found that while bacterial growth was consistently higher on low than high melanin necromass, the CAZyme-encoding gene expression response of the three species was similar between the two necromass types. Interestingly, this trend was not shared for genes encoding nitrogen utilization, which varied in C. pinensis and S. marcescens during growth on high vs low melanin necromass. Additionally, this study tested the metabolic capabilities of these bacterial species to grow on a diversity of C and N sources and found that the three bacteria have substantially different utilization patterns. Collectively, our data suggest that as necromass changes chemically over the course of degradation, certain bacterial species are favored based on their differential metabolic capacities.IMPORTANCEFungal necromass is a major component of the carbon (C) in soils as well as an important source of nitrogen (N) for plant and microbial growth. Bacteria associated with necromass represent a distinct subset of the soil microbiome and characterizing their functional capacities is the critical next step toward understanding how they influence necromass turnover. This is particularly important for necromass varying in melanin content, which has been observed to control the rate of necromass decomposition across a variety of ecosystems. Here we assessed the gene expression of three necromass-degrading bacteria grown on low or high melanin necromass and characterized their metabolic capacities to grow on different C and N substrates. These transcriptomic and metabolic studies provide the first steps toward assessing the physiological relevance of up-regulated CAZyme-encoding genes in necromass decomposition and provide foundational data for generating a predictive model of the molecular mechanisms underpinning necromass decomposition by soil bacteria.

Genotype and Nitrogen Source Influence Drought Stress Response in Oil Palm Seedlings

Genotype and Nitrogen Source Influence Drought Stress Response in Oil Palm Seedlings

High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen.

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludis can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain.IMPORTANCEArchaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis. RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.

Nitrogen signaling factor triggers a respiration-like gene expression program in fission yeast.

Microbes have evolved intricate communication systems that enable individual cells of a population to send and receive signals in response to changes in their immediate environment. In the fission yeast Schizosaccharomyces pombe, the oxylipin nitrogen signaling factor (NSF) is part of such communication system, which functions to regulate the usage of different nitrogen sources. Yet, the pathways and mechanisms by which NSF acts are poorly understood. Here, we show that NSF physically interacts with the mitochondrial sulfide:quinone oxidoreductase Hmt2 and that it prompts a change from a fermentation- to a respiration-like gene expression program without any change in the carbon source. Our results suggest that NSF activity is not restricted to nitrogen metabolism alone and that it could function as a rheostat to prepare a population of S. pombe cells for an imminent shortage of their preferred nutrients.

Enhanced polyhydroxyalkanoate production from Mesobacillus aurentius: Statistical optimization, characterization and industrial application

Synthetic plastics pose a major environmental threat and it is necessary to produce an alternative biopolymer. In the current study, the production of polyhydroxybutyrate (PHB) by Mesobacillus aurentius was enhanced using Response surface methodology (Box–Behnken design). This study explores the potential of aquabiofloc systems as a source of polyhydroxyalkanoates (PHB)-producing bacteria. The optimized medium conditions, as determined by Response Surface Methodology (RSM), included 18.68 g of sucrose, 4.0 g of yeast, an incubation period of 69.57 h, and a pH of 7.1. The ANOVA results revealed that the model developed for predicting PHA yield was highly significant (p < 0.05). The predicted PHA yield was 63.12%, while the experimental yield was 65.35%. The maximum production of PHA was obtained with sucrose and yeast as carbon and nitrogen sources. The extracted polymer was characterized using UV, FTIR, 1H NMR, and SEM-EDAX analysis confirming the polymer to be PHB. The thermal stability of the produced PHA showed degradation temperatures ranging from 310 °C. The mechanical properties of the extracted PHA were also assessed, demonstrating tensile strength and viscosity of 22.4 MPa and 1.23 MPa. respectively. The antimicrobial activity of the produced PHA was evaluated, demonstrating significant inhibitory effects against both Gram-positive and Gram-negative bacterial strains as well as fungal strains. The Cytotoxicity assessment in HepG2 cells indicated that PHB is less toxic in nature. The findings highlight the promising role of marine bacteria, Mesobacillus aurentius, in the development of environmentally friendly biopolymers. This bacterium represents a novel candidate for PHB production, offering a potential alternative to petroleum-based plastics.

Nitrogen-doped mesoporous carbon as metal-free catalysts for the aerobic oxidation of alcohols

The selective oxidation of alcohols to carbonyl compounds is a crucial reaction in organic chemistry. In this study, we report the synthesis of nitrogen-doped mesoporous carbon material by hard template method using biomass derivative sucrose and melamine as carbon and nitrogen sources and silica sol as template agent. Using molecular oxygen as the oxidant, the obtained NC-900 exhibits excellent selectivity in the oxidation of primary alcohols to the corresponding aldehydes without generating over-oxidized acid. For example, under optimal reaction conditions, 4-methoxybenzyl alcohol is almost quantitatively converted to 4-methoxybenzaldehyde. The catalytic system showed robust tolerance to sensitive functional groups and demonstrated favorable reaction effects in gram-scale experiments and multi-step, one-pot reaction sequences. The catalyst was reused for 5 times, and its catalytic activity did not decrease significantly. Hammett correlation studies indicated that the reaction favored substrates with electron-donating substituents. Characterization and experimental results indicate that the synergistic effect of graphitic N and mesoporous structure plays a pivotal role in enhancing the catalytic activity of NC-900 in this transformation. The mechanism study shows that the reaction involves free radical process.

Engineering the xylose metabolism of Saccharomyces cerevisiae for ethanol and single cell protein bioconversion

Xylose isomerase (XI) pathway has been widely employed to enable Saccharomyces cerevisiae to convert xylose and glucose into commercially feasible lignocellulosic ethanol products. Nevertheless, studies about the effect of different promoters to the expression of xylA are lacking. Therefore, five strains with ADH1, GAPDH, PDC1, PGK, TEF1 promoters were constructed. Among them, S. cerevisiae INVSc1/pHM368-PADH1-xylA generated with xylA driven by ADH1 promoter displayed the highest xylose utilization rate (approximately 19.98 %) using xylose as the only carbon source. With 4 g/L glucose and 10 g/L xylose as the carbon sources, the xylose utilization rate was 60.04 %. Moreover, the utilization rate increased to 64.04 % with fermentation temperature elevated from 28 °C to 30 °C and reached 83.09 % with peptone and yeast extract as the nitrogen sources. The ethanol titer reached 1.74 g/L with a yield of 0.38 g/g sugar under this condition. In Comparison with direct fermentation, the single cell protein (SCP) was 1.27-fold higher during aerobic fed-batch fermentation. Furthermore, INVSc1/pHM368-PADH1-xylA attains high ethanol productivities and yields by converting glucose and xylose from non-detoxified bagasse hydrolysates as carbon sources. The results extend our understanding of the xylose metabolism of S. cerevisiae and provide a platform for biomass conversion to ethanol and SCP, hence paving the way for the development of a more economical and sustainable approach to co-fermentation performance and capabilities for future engineering.

A dynamic model for predicting biomass and phycocyanin yields in Arthrospira (Spirulina) platensis: A guidance for effective batch cultivation

C-phycocyanin (C-PC) is a highly valuable bioproduct from the cyanobacterium Arthrospira platensis. A crucial factor affecting growth and C-PC production yield is nitrogen nutrients. In this work, an ODE-based dynamic model was constructed to simulate the effect of ammonium concentrations in a batch system on cyanobacterial growth and C-PC production. The model included dynamic regulation of the ammonium transporter and key enzymes involved in the nitrogen assimilation pathway. The prediction of C-PC production, cyanobacterial growth, and remaining ammonium concentration over 24 h strongly correlated with experimental data. Furthermore, the model was able to capture the response of genes involved in ammonium assimilation and C-PC production, as well as the primary metabolites. The dynamic interplay among ammonium, glutamine, and glutamate levels reflects the complexity of nitrogen metabolism in regulating the transcription of genes involved in ammonium uptake, assimilation, and C-PC synthesis and degradation, thus highlighting the cellular response to nitrogen stress. These findings provide a foundation for understanding these biological processes and offer a potential tool for further exploring the complex relationship between nitrogen availability and C-PC accumulation in A. platensis C1 using ammonium as a nitrogen source.

Characterization of the extracellular proteases from Bacillus inaquosorum strain E1-8 and its application in the preparation of hydrolysates from plant and animal proteins with antioxidant, antifreeze and anti-browning properties.

Bacillus inaquosorum strains is widely recognized for their plant-growth-promoting and biocontrol capabilities, yet their roles in protease production remain unclear. The present study aimed to comprehensively assess the protease-producing performance of B. inaquosorum strain E1-8, at the same time as exploring the novel application of agricultural Bacillus proteases in the preparation of protein hydrolysates for fresh-cut fruits preservation. First, genomic sequencing revealed the diversity of E1-8 proteases, indicating 15 putative extracellular proteases. Subsequently, the fermentation conditions for E1-8 protease production were optimized, with sweet potato powder and soybean meal identified as the most suitable carbon and nitrogen sources, respectively, resulting in a maximum protease activity of 321.48 U mL-1. Upon culturing the strain under these optimized conditions, only an S8 family serine protease and an M48 family metalloprotease were revealed by secretomic analysis and protease inhibitor assays. Additionally, the optimal protease conditions for generating protein hydrolysates from soy, pea, fish and porcine proteins were determined. The molecular weight of the hydrolysates primarily ranged from 2000 to 180 Da, with a total of 17 amino acids identified. The application of these hydrolysates demonstrated a 2,2-diphenyl-1-picrylhydrazyl (i.e. DPPH) scavenging activity ranging from 58.64% to 84.12%, significantly reducing of the melting peaks and the freezing points. Furthermore, the browning index of apple slices stored at 4 °C decreased by 14.81% to 22.15% on the second day, and similar effects were observed in fresh-cut banana stored at 4 °C for 7 days. The protein hydrolysates obtained exhibit remarkable antioxidant, antifreeze and anti-browning properties for fresh-cut fruits. © 2024 Society of Chemical Industry.

Field Study and Numerical Modeling to Assess the Impact of On-Site Septic Systems on Groundwater Quality of Jeju Island, South Korea

Septic-derived nitrogen (N) sources have harmful effects on water resources, humans, and ecosystems in several countries. On Jeju Island, South Korea, the rapid increase in personal sewage treatment facilities (PSTFs, also known as on-site septic systems) raises concerns regarding the deterioration of groundwater quality, as groundwater is the sole water resource on the island. Therefore, this study employed a field study and numerical modeling to assess the impact of PSTF effluents on groundwater quality in the Jocheon area of northeastern Jeju. Water quality analysis revealed that the total nitrogen (T-N) concentrations in the effluent exceeded the effluent standards (75–92% PSTFs). The numerical model simulated the transport of N species, showing limited NH4+ and NO2− plume migration near the surface due to nitrification and adsorption. However, NO3− concentrations increased and stabilized over time, leaching on the water table with higher levels in lowland areas and clustered PSTFs. The predictive model estimated a 79% reduction in NO3− leaching when the effluents followed standards, indicating the necessity of effective PSTF management. This study highlights the importance of managing improperly operated septic systems to mitigate groundwater contamination based on an understanding of the behavior of N species in subsurface hydrologic systems.

Effect of NH4Cl supplementation on growth, photosynthesis, and triacylglycerol content in Chlamydomonas reinhardtii under mixotrophic cultivation.

Ammonium chloride (NH4Cl) is one of the nitrogen sources for microalgal cultivation. An excessive amounts of NH4Cl are toxic for microalgae. However, combining mixotrophic conditions and excessive quantities of NH4Cl positively affects microalgal biomass and lipid production. In this study, we investigated the impact of NH4Cl on the growth, biomass, and triglyceride (TAG) content of the green microalga Chlamydomonas reinhardtii especially under mixotrophic conditions. Under photoautotrophic conditions (without organic carbon supplementation), adding 25 mM NH4Cl had no significant effect on microalgal growth or TAG content. However, under mixotrophic condition (with acetate supplementation), NH4Cl interfered with microalgal growth while inducing TAG content. To explore these effects further, we conducted a two-step cultivation process and found that NH4Cl reduced microalgal growth, but induced total lipid and TAG content, especially after 4-day cultivation. The photosynthesis performances showed that NH4Cl completely inhibited oxygen evolution on day 4. However, NH4Cl slightly reduced the Fv/Fm ratio indicating that the NH4Cl supplementation directly affects microalgal photosynthesis. To investigate the TAG induction effect by NH4Cl, we compared the protein expression profiles of microalgae grown mixotrophically with and without 25 mM NH4Cl using a proteomics approach. This analysis identified 1782 proteins, with putative acetate uptake transporter GFY5 and acyl-coenzyme A oxidase being overexpressed in the NH4Cl-treated group. These findings suggested that NH4Cl supplementation may stimulate acetate utilization and fatty acid synthesis pathways in microalgae cells. Our study indicated that NH4Cl supplementation can induce microalgal biomass and lipid production, particularly when combined with mixotrophic conditions.