Nitrogen Limitation Research Articles

Response of soil micro-food web to nutrient limitation along a subtropical forest restoration

Forest ecosystem productivity and function is strongly influenced by the interaction between soil organisms and their resource use that can be impeded by an imbalance of ecological stoichiometry. Soil microorganisms are known to have an important role in biogeochemical cycling which is strongly influenced by ecological stoichiometry. However, there is limited understanding of how soil micro-food web respond to stoichiometric imbalances during forest restoration. Here, we investigated the effect of forest restoration on soil physio-chemical properties and the structure and function of soil micro-food web along a chronosequence of transformation stages: (i) early stage monoculture plantation of Chinese fir (Cunninghamia lanceolata) comprised of three age classes (5, 10 and 20 years); (ii) mid-stage conifer-broadleaved mixed forest; and (iii) late-stage mixed species broadleaved forest in south China. Results showed that forest restoration from C. lanceolata monocultures to mixed species broadleaved forest significantly increased soil organic carbon and total nitrogen. Soil bacteria, fungi, protists and nematodes abundance increased and the co-occurrence networks of soil biota became more complex and stable along the restoration chronosequence. In contrast, soil nitrogen and phosphorus limitations, particularly phosphorus limitation, increased along the chronosequence. In addition, soil exoenzyme activity suggested that the microbial investment in resource acquisition shifted from C- to nutrient-acquiring enzymes from the earlier to the later restoration stages. Availability of soil resources (e.g., dissolved organic carbon, ammonium, and available phosphate) appeared to have an important role in regulating soil food web composition, structure and stability during forest restoration. We conclude that nutrient limitation, particularly phosphorus limitation, likely has an important role in determining the stability of soil food webs during forest restoration. These findings contribute to our understanding of the relationships between soil nutrient limitation and soil micro-food web, and have implications for carbon sequestration through forest restoration and management in southern China.

Yeast Lipids from Crude Glycerol Media and Utilization of Lipid Fermentation Wastewater as Maceration Water in Cultures of Edible and Medicinal Mushrooms

Four wild “red” yeast strains (Rhodosporidium kratochvilovae FMCC Y70, R. toruloides NRRL Y-27013, R. toruloides NRRL Y-17902 and R. toruloides NRRL Y-6985) were cultured in shake flasks on industrial glycerol at an initial substrate (Gly0) concentration ≈ 50 g/L under nitrogen limitation. Strains NRRL Y-27013, NRRL Y-17902 and NRRL Y-6985 presented appreciable dry cell weight (DCW) and lipid synthesis (DCW up to 18–19 g/L containing lipids in quantities ≈ 47%, w/w). Strains NRRL Y-27013 and NRRL Y-6985 were further tested in higher Gly0 concentrations (≈90 g/L and ≈110 g/L) with the same initial nitrogen quantity as in the first (“screening”) experiment. Both strains, despite the high Gly0 concentrations and C/N ratios (up to 120 moles/moles) imposed, presented significant DCW production (up to c. 29.0–29.5 g/L). Yeast biomass contained significant lipid (42–43%, w/w) and endopolysaccharide (up to 42%, w/w) quantities. Both lipids and endopolysaccharide quantities (in % w/w) noticeably increased as a response to the imposed nitrogen limitation. Lipids containing mainly oleic and palmitic acids constituted ideal candidates for biodiesel synthesis. Thereafter, the wastewaters derived from the lipid production process (lipid fermentation wastewaters—LFWs) were used as maceration waters in cultivations of edible and medicinal fungi, where novel (non-conventional) substrates were used in the performed cultures. CW (coffee residue + wheat straw), CB (coffee residue + beech wood shavings), OW (olive crop + wheat straw), OB (olive crop + beech wood shavings), RW (rice husk + wheat straw) and RB (rice husk + beech wood shavings) were soaked/sprayed with LFWs or tap water and utilized in the cultivation of Pleurotus, Ganoderma and Lentinula mushrooms. The impact of LFWs on the mycelial growth rate (mm/d) and biomass production was evaluated. The results show that regardless of the wetting method, the highest growth rates (6.2–6.6 mm/d) were noticed on RW and RB for Pleurotus eryngii and Ganoderma resinaceum, on OW, OB and RW for Ganoderma applanatum and on RW, OW and OB for Lentinula edodes. Nevertheless, high biomass production was obtained on substrates soaked with LFWs for Pleurotus ostreatus (RW: 443 mg/g d.w.), L. edodes (RB: 238 mg/g d.w.) and Ganoderma lucidum (RW: 450 mg/g d.w.). Overall, this study demonstrates the possibility of the industrial conversion of low-value agro-waste to mycelial mass and eventually to important food products.

Impact of dust deposition on the growth of marine autotrophic and heterotrophic microorganisms: Evidence from the South China Sea

Aeolian dust can provide nutrients for the ocean and affect the growth of phytoplankton. However, the impacts of dust deposition on autotrophic and heterotrophic microorganisms have rarely been studied. In this study, we conducted two microcosm experiments in the low-nutrient and low-chlorophyll environment of the South China Sea and found that dust did not stimulate the abundance of autotrophic and heterotrophic microorganisms. Our results show that dust contains most of the unreacted iron-bearing minerals, and thus provides limited bioavailable iron and nitrogen for bacterioplankton and phytoplankton growth. These results elucidate the overlooked impacts of the properties of the iron-bearing minerals in aeolian dust on microbial communities, which may play an important role in marine ecosystems and climate change.

Mixed sowing of Feed rape and Vicia villosa can substitute nitrogen fertilizer to improve soil multifunctionality in the Hetao irrigation District

Soil multifunctionality (SMF) and crop production of continuous green manure returning is crucial for sustainable agricultural development. Multiple cropping of green manure after wheat harvest is a typical planting pattern in the Hetao Irrigation District, and maybe substitute nitrogen (N) fertilizer. However, information is scarce about how the green manure affected SMF and crop production under limited nitrogen inputs. A 7-year field experiment was conducted to investigate the effects of green manure types (Feed rape (FR), Vicia villosa (VV), Feed rape, and Vicia villosa mixed sowing (FR + VV)) on soil properties, enzyme activities, SMF, and rotated wheat yield under different N application rates (150 kg N ha−1, N150; no nitrogen, N0). The results showed that FR and FR + VV increased soil active carbon (C) and N fractions by 4.0 %-499.3 % and 11.3 %-147.8 % compared to VV, respectively. Enzyme activities with FR + VV were significantly increased by 13.7 %-82.9 % compared to FR and VV. SMF and CCI (carbon cycling index) were significantly affected by N fertilizer and green manure types, with N0 higher than N150 by two times and FR + VV higher than FR by 1.4–4.3 times and VV by 1.0–6.6 times in 0–20 cm soil layer. Spring wheat yield was significantly affected by N fertilizer and green manure types, with N0 significantly higher than N150 by 5.8 % and FR and FR + VV higher than VV by 8.0 % and 12.5 % under N150, respectively. Partial least squares path model detected that returning of multiple green manure had a significant positive and indirect effect on SMF through enhancing soil enzyme activities, and ultimately contribute to the improvement in wheat yield. In conclusion, the mixed sowing of FR and VV increased soil active C and N fractions and associated enzyme activities, and has the potential to improve the SMF and rotated wheat yield, thereby providing an effective approach to reduce N fertilizer.

Small protein mediates inhibition of ammonium transport in Methanosarcina mazei-an ancient mechanism?

In the past decade, small open reading frames (sORFs) coding for proteins less than 70 amino acids (aa) in length have moved into the focus of science. sORFs and the corresponding small proteins have been recently identified in all three domains of life. However, the majority of small proteins remain functionally uncharacterized. While several bacterial small proteins have already been described, the number of identified and functionally characterized small proteins in archaea is still limited. In this study, we have discovered that the small protein 36 (sP36), which consists of only 61 aa, plays a critical role in regulating nitrogen metabolism in Methanosarcina mazei. The absence of sP36 significantly delays the growth of M. mazei when transitioning from nitrogen limitation to nitrogen sufficiency, as compared to the wild type. Through our in vivo experiments, we have observed that during nitrogen limitation, sP36 is dispersed throughout the cytoplasm; however, upon shifting the cells to nitrogen sufficiency, it relocates to the cytoplasmic membrane. Furthermore, an in vitro biochemical analysis clearly showed that sP36 interacts with high affinity with the ammonium transporter AmtB1 present in the cytoplasmic membrane during nitrogen limitation as well as with the PII-like protein GlnK1. Moreover, the in vivo GlnK1 interaction with AmtB1 due to nitrogen upshifts requires the presence of sP36. Based on our findings, we propose that in response to an ammonium upshift, sP36 targets the ammonium transporter AmtB1 and inhibits its activity by mediating the interaction with GlnK1. IMPORTANCE Small proteins containing fewer than 70 amino acids, which were previously disregarded due to computational prediction and biochemical detection challenges, have gained increased attention in the scientific community in recent years. However, the number of functionally characterized small proteins, especially in archaea, is still limited. Here, by using biochemical and genetic approaches, we demonstrate a crucial role of the small protein sP36 in the nitrogen metabolism of M. mazei, which modulates the ammonium transporter AmtB1 according to nitrogen availability. This modulation might represent an ancient archaeal mechanism of AmtB1 inhibition, in contrast to the well-studied uridylylation-dependent regulation in bacteria.

Herbivores influence biogeochemical processes by altering litter quality and quantity in a subarctic wetland

Global change drivers that modify the quality and quantity of litter inputs to soil affect greenhouse gas fluxes, and thereby constitute a feedback to climate change. Carbon cycling in the Yukon–Kuskokwim (Y–K) River Delta, a subarctic wetland system, is influenced by landscape variations in litter quality and quantity generated by herbivores (migratory birds) that create ‘grazing lawns’ of short stature, nitrogen-rich vegetation. To identify the mechanisms by which these changes in litter inputs affect soil carbon balance, we independently manipulated qualities and quantities of litter representative of levels found in the Y–K Delta in a fully factorial microcosm experiment. We measured CO2 fluxes from these microcosms weekly. To help us identify how litter inputs influenced greenhouse gas fluxes, we sequenced soil fungal and bacterial communities, and measured soil microbial biomass carbon, dissolved carbon, inorganic nitrogen, and enzyme activity. We found that positive correlations between litter input quantity and CO2 flux were dependent upon litter type, due to differences in litter stoichiometry and changes to the structure of decomposer communities, especially the soil fungi. These community shifts were particularly pronounced when litter was added in the form of herbivore feces, and in litter input treatments that induced nitrogen limitation (i.e., senesced litter). The sensitivity of carbon cycling to litter quality and quantity in this system demonstrates that herbivores can strongly impact greenhouse gas fluxes through their influence on plant growth and tissue chemistry.Graphical abstract

Coupled carbon and nitrogen cycling regulates the cnidarian–algal symbiosis

Efficient nutrient recycling underpins the ecological success of cnidarian-algal symbioses in oligotrophic waters. In these symbioses, nitrogen limitation restricts the growth of algal endosymbionts in hospite and stimulates their release of photosynthates to the cnidarian host. However, the mechanisms controlling nitrogen availability and their role in symbiosis regulation remain poorly understood. Here, we studied the metabolic regulation of symbiotic nitrogen cycling in the sea anemone Aiptasia by experimentally altering labile carbon availability in a series of experiments. Combining 13C and 15N stable isotope labeling experiments with physiological analyses and NanoSIMS imaging, we show that the competition for environmental ammonium between the host and its algal symbionts is regulated by labile carbon availability. Light regimes optimal for algal photosynthesis increase carbon availability in the holobiont and stimulate nitrogen assimilation in the host metabolism. Consequently, algal symbiont densities are lowest under optimal environmental conditions and increase toward the lower and upper light tolerance limits of the symbiosis. This metabolic regulation promotes efficient carbon recycling in a stable symbiosis across a wide range of environmental conditions. Yet, the dependence on resource competition may favor parasitic interactions, explaining the instability of the cnidarian-algal symbiosis as environmental conditions in the Anthropocene shift towards its tolerance limits.

Terrestrial Trentepohlia sp. (Ulvophyceae) from alpine and coastal collection sites show strong desiccation tolerance and broad light and temperature adaptation.

For the present study, we collected the Ulvophyceae species Trentepohlia aurea from limestone rock near Berchtesgaden, Germany, and the closely related taxa T. umbrina from Tilia cordata tree bark and T. jolithus from concrete wall both in Rostock, Germany. Freshly sampled material stained with Auramine O, DIOC6, and FM 1-43 showed an intact physiological status. Cell walls were depicted with calcofluor white and Carbotrace. When subjected to three repeated and controlled cycles of desiccation over silica gel (~ 10% relative humidity) followed by rehydration, T. aurea recovered about 50% of the initial photosynthetic yield of photosystem II (YII). In contrast, T. umbrina and T. jolithus recovered to 100% of the initial YII. HPLC and GC analysis of compatible solutes found highest proportions of erythritol in T. umbrina and mannitol/arabitol in T. jolithus. The lowest total compatible solute concentrations were detected in T. aurea, while the C/N ratio was highest in this species, indicative of nitrogen limitation. The prominent orange to red coloration of all Trentepohlia was due to extremely high carotenoid to Chl a ratio (15.9 in T. jolithus, 7.8 in T. aurea, and 6.6. in T. umbrina). Photosynthetic oxygen production was positive up to ~ 1500µmol photons m-2s-1 with the highest Pmax and alpha values in T. aurea. All strains showed a broad temperature tolerance with optima for gross photosynthesis between 20 and 35°C. The presented data suggest that all investigated Trentepohlia species are well adapted to their terrestrial lifestyle on exposed to sunlight on a vertical substrate with little water holding capacity. Nevertheless, the three Trentepohlia species differed concerning their desiccation tolerance and compatible solute concentrations. The lower compatible solute contents in T. aurea explain the incomplete recovery of YII after rehydration.

Control of glnA (glutamine synthetase) expression by urea in non-pathogenic and uropathogenic Escherichia coli.

The bacteria that cause urinary tract infections often become resistant to antibiotic treatment, and genes expressed during an infection could suggest non-antibiotic targets. During growth in urine, glnA (specifying glutamine synthetase) expression is high, but our results show that urea induces glnA expression independent of the regulation that responds to nitrogen limitation. Although our results suggest that glnA is an unlikely target for therapy because of variation in urinary components between individuals, our analysis of glnA expression in urine-like environments has revealed previously undescribed layers of regulation. In other words, regulatory mechanisms that are discovered in a laboratory environment do not necessarily operate in the same way in nature.

Wax Ester and Triacylglycerol Production in Acinetobacter baumannii: Role in Osmostress Protection, Reactive Oxygen Species, and Antibiotic Sensitivity.

Wax esters (WEs) are neutral lipids that are produced by many different bacteria as potential carbon and energy storage compounds. Comparatively little is known about the role of WE in pathogenic bacteria. The opportunistic pathogen Acinetobacter baumannii is a major cause of hospital-acquired infections worldwide. Salt and desiccation resistance foster A. baumannii infections such as urinary tract infections and allow for reinfection when bacteria are taken up from dry surfaces in the hospital environment. Here we report on WE and triacylglycerol (TAG) production in A. baumannii as a response to nitrogen limitation and high salt stress. Fatty acids and fatty alcohols with chain lengths of C16 and C18 were identified as the most prominent WE constituents. We identified the terminal key enzyme of WE biosynthesis, the bifunctional wax ester synthase/acylCoA:diacylglycerol acyltransferase (WS/DGAT) encoded by the wax/dgat gene, and demonstrated that transcription of wax/dgat and production of WS/DGAT are independent of the nitrogen concentration. A Δwax/dgat mutant was impaired in growth in the presence of high salt concentration and was more sensitive to imipenem and reactive oxygen species.

Inter-Month Nutrients Dynamic and Plant Growth in Calamagrostis angustifolia Community and Soil after Different Burning Seasons

Presently, as human activity and climate warming gradually increase, straw burning leads to more accidental burning in neighbouring wetlands, which threatens wetland carbon stores. Plants are important carbon fixers in wetlands, converting carbon dioxide to biomass through photosynthesis and releasing carbon into the soil as plants die off. Nitrogen and phosphorus limitation in wetlands is a key factor affecting plant growth, and different burning seasons have different effects on mitigating this limitation. To further elucidate the effects of nitrogen and phosphorus distribution on wetland inter-month nutrient dynamics after different burning seasons, we selected a Calamagrostis angustifolia wetland in the Sanjiang Plain that was burned in spring and autumn, respectively, and conducted a monthly survey from May to September. We found that the leaf nitrogen content in September at spring burning sites was 3.59 ± 2.69 g/kg, which was significantly lower than that in July, while the difference at the unburned sites was only 0.60 ± 3.72 g/kg, and after the autumn burning, soil nitrogen and phosphorus contents remained higher than at the unburned sites in August, being 0.55 ± 1.74 g/kg and 0.06 ± 0.12 g/kg, respectively. Our results indicate that spring burning immediately increased the nitrogen and phosphorus contents in soil and plants but that these effects only lasted for a short time, until June. In comparison, autumn burning had a long-term effect on soil nitrogen and phosphorus levels and significantly increased the aboveground biomass. Thus, we recommend that conducting autumn burning before the commencement of agricultural burning not only reduces combustible accumulation to prevent fires but also promotes nitrogen and phosphorus cycling in wetlands, and the increase in plant biomass after autumn burning also enhances the carbon fixation capacity of the wetland.

Late nitrogen fertilization increases biomass of cotton bolls by reinforcing source[sbnd]sink performance

The decrease in reproductive organ biomass is a principal constraint for cotton yield under conditions of limited nitrogen (N). We aimed to alleviate this challenge by reasoning N applications in order to exploit the photosynthetic potential and improve the distribution of photosynthates. Cotton crops were grown in a two-year field experiment (2019–2020) under N-reduced cultivation (23.9 ×104 plants ha−1, 76 cm row spacing, and applied 240 kg N ha−1), N fertigated with five ratios at squaring (SQ): flowering to peak boll (FPB): late peak boll (LPB), i.e., 4:6:0 (N460), 3:6:1 (N361), 2:6:2 (N262, Control), 1:6:3 (N163) and 0:6:4 (N064). Impacts of N treatment (ratio) were investigated for leaf area, chlorophyll content, photosynthetic characteristics, photosynthetic N use efficiency (PNUE), sink capacity characteristics, and biomass accumulation. Correlations between traits were also analyzed. Compared with conventional N262, N064 increased reproduction organs biomass by 10.1–10.3%. Furthermore, during the LPB to boll opening stage, N064 increased fruit branch leaf area by 1.4–26.8%, chlorophyll content by 6.4–27.6%, net photosynthetic rate by 15.1–35.9%, actual photosynthetic efficiency by 14.4–41.6%, photochemical quenching by 18.0–57.7%, PNUE by 15.7–41.0%, and sink capacity by 6.7–10.5%; while it decreased foliage branch leaf area by 13.1–22.4% and chlorophyll a/b by 6.1–10.2%. Overall, late N fertilization was an effective strategy to (1) retain post-flowering photosynthetic capacity by developing more fruit branch leaf area with higher chlorophyll content and lower chlorophyll a/b, (2) optimize sourcesink relationship by increasing boll capacity, and (3) increase biomass in reproductive organs, thus enhancing sustainable and efficient production of cotton under N-reduced cultivation in arid regions.

The Root-Colonizing Endophyte Piriformospora indica Supports Nitrogen-Starved Arabidopsis thaliana Seedlings with Nitrogen Metabolites.

The root-colonizing endophytic fungus Piriformospora indica promotes the root and shoot growth of its host plants. We show that the growth promotion of Arabidopsis thaliana leaves is abolished when the seedlings are grown on media with nitrogen (N) limitation. The fungus neither stimulated the total N content nor did it promote 15NO3- uptake from agar plates to the leaves of the host under N-sufficient or N-limiting conditions. However, when the roots were co-cultivated with 15N-labelled P. indica, more labels were detected in the leaves of N-starved host plants but not in plants supplied with sufficient N. Amino acid and primary metabolite profiles, as well as the expression analyses of N metabolite transporter genes suggest that the fungus alleviates the adaptation of its host from the N limitation condition. P. indica alters the expression of transporter genes, which participate in the relocation of NO3-, NH4+ and N metabolites from the roots to the leaves under N limitation. We propose that P. indica participates in the plant's metabolomic adaptation against N limitation by delivering reduced N metabolites to the host, thus alleviating metabolic N starvation responses and reprogramming the expression of N metabolism-related genes.

Competition-induced downregulation of symbiotic nitrogen fixation.

Controlled experiments at the level of individual plants show that legume species use different strategies for the regulation of symbiotic dinitrogen fixation in response to nitrogen availability. These strategies were suggested to improve legume fitness in the context of the plant community, although rarely studied at this level. We evaluated how nitrogen availability and conspecific vs heterospecific interactions influenced the strategy of regulation of nitrogen fixation. We grew two species of herbaceous legumes representing two different strategies of regulation without interaction, under treatments of deficient and sufficient nitrogen availability, with conspecific or heterospecific interaction. We found that Hymenocarpus circinnatus maintained a facultative strategy of downregulating nitrogen fixation when nitrogen was available under both con- and heterospecific interactions, as was also found for this species when grown alone. Vicia palaestina also downregulated nitrogen fixation under both con- and heterospecific interactions but did not regulate fixation when grown alone. Our results showed that under nitrogen limitation, interaction with a neighboring plant reduced fitness, reflecting a competitive effect. Our findings suggest that when interacting with other plants, downregulation of nitrogen fixation is more likely, therefore reducing the energetic cost of fixation, and improving plant performance in competitive ecological communities, especially when nitrogen is available.

Proteomic response of Phaeocystis globosa to nitrogen limitation

Proteomic response of Phaeocystis globosa to nitrogen limitation

Tracking the sources of Leptospira and nutrient flows in two urban watersheds of Puerto Rico.

This study investigated the relationship between nutrient levels, source of fecal contamination, and pathogenic Leptospira in Puerto Rico's northern coast and San Juan Bay Estuary (SJBE) aquatic ecosystems. Microbial source tracking (MST) was also used to investigate the connections between sources of feces contamination and the presence of Leptospira. Eighty-seven water samples were collected during the June (n=44) and August (n=43) in 2020. To quantify phosphorus and nitrogen concentrations, standard USEPA protocols were utilized, specifically Methods 365.4 for total and dissolved phosphorus, 351.2 for total Kjeldahl nitrogen and ammonium, and 353.2 for nitrate. Lipl32 gene-specific quantitative polymerase chain reaction (qPCR) was used to detect the presence of Leptospira. Human (HF183), canine (BacCan-UCD), and equine (HoF597) MST assays were utilized to trace the origins of fecal contamination. Forty one percent of the locations exceeded Puerto Rico's authorized total phosphorus limit of 160 g L-1, while 34% exceeded the total nitrogen limit of 1700 g L-1. Nearly half of the streams examined are affected by eutrophication. The MST analysis identified human and canine feces as the most prevalent contaminants, affecting approximately 50% of the sites. In addition, Leptospira was detected in 32% of the June samples. There was a significant correlation (r = 0.79) between the incidence of pathogenic Leptospira and the human bacterial marker (HF183). This study illuminates the central role of anthropogenic inputs in nutrient enrichment and pathogen proliferation in Puerto Rico's aquatic ecosystems.

The Role of Heterocysts in Cyanotoxin Production during Nitrogen Limitation.

Cyanobacteria harmful algal blooms (cyanoHABs) can have impacts on human health, aquatic ecosystems, and the economy. Nutrient management is an important mitigation and even remediation strategy. In this work, the paralytic shellfish toxin (PST)-producing Aphanizomenon (Aphan.) flos-aquae (Linnaeus) Ralfs ex Bornet & Flahault (now identified as Aphan. sp.) single filament isolate NH-5 was grown in P-depleted media, N-depleted media, and complete BG-11 media. Growth and heterocyst and vegetative cells were monitored using dry weight and cell counts. Ultrasonication was used to separate heterocysts from vegetative cells. HPLC-FLD with post-column derivatization was used to determine the saxitoxin (STX) and neosaxitoxin (NEOSTX) concentration per cell. Aphan. sp. NH-5 biomass was lower in the P-depleted media than in the N-depleted media and the control, though higher heterocyst counts were detected in the N-depleted media. The heterocyst toxin concentration was significantly higher compared to the vegetative cells for the N-depleted media, control, and P-depleted media. However, no significant differences were found among all preparations with regard to the STX-to-NEOSTX ratio. We conclude that N limitation induced higher heterocyst numbers and that N fixation activity is a factor behind the increase in the STX and NEOSTX production of Aphan. sp. NH-5.

Assessment of Eutrophication Characteristics and Evaluation of the First-Generation Eutrophication Model in the Nearshore Waters of Shantou City

Based on the water quality monitoring results from 2002 to 2018 in the coastal waters of Shantou City, this study used eight methods, including the single-factor index method, eutrophication index method, nutrient status quality method, comprehensive index method, organic pollution index method, nitrogen–phosphorus ratio method, potential eutrophication evaluation, and dissolved oxygen saturation parameter method, to evaluate the eutrophication and organic pollution status of the area. The applicability of the first-generation eutrophication model was assessed from the perspective of red tide occurrence, and a comparison was made among the various evaluation methods. The results indicate that the coastal waters of the study area were mostly in a phosphorus-limited state, with a few periods of nutrient deficiency and a very small portion of time in a moderately nutritious state with both nitrogen and phosphorus limitations. The overall N/P ratio showed an increasing trend primarily due to the gradual increase in inorganic nitrogen content. In terms of evaluating eutrophication based on red tide symptoms, the comprehensive index method showed better performance in reflecting eutrophication conditions, while the eutrophication index method and dissolved oxygen saturation parameter method highlighted problematic areas but often exaggerated their extent. The nutrient status quality method and organic pollution index method did not provide distinctive indications and could not verify red tide symptoms. The nitrogen–phosphorus ratio method and potential eutrophication evaluation could reflect certain nutrient structures and eutrophication characteristics but also failed to verify red tide symptoms. Furthermore, this study found a certain correlation between the occurrence of red tide and the ratio of nitrate nitrogen to ammonia nitrogen, which consistently ranged from 1.15 to 1.94 during red tide events. Therefore, whether this ratio can serve as a characteristic value for red tide occurrence and be used for early warning and monitoring still requires further validation.

Role of AMP Deaminase in Mucor circinelloides: Implications for Nitrogen Utilization and Lipid Biosynthesis.

Lipid accumulation in oleaginous organisms is initiated by AMP deaminase (AMPD) after nitrogen depletion because it mediates the concentration of intracellular adenosine monophosphate (AMP). However, the role of AMPD in lipogenesis in the oleaginous fungus Mucor circinelloides is largely unknown. Therefore, we identified the genes (ampd1 and ampd2) encoding AMPD and investigated the role of AMPD in lipid synthesis in this fungus by overexpressing and deleting ampd genes. Deletion of ampd1 and ampd2 caused 21 and 28% increments in lipid contents under N-limited conditions, respectively. These increases were correlated with the activation of enzymes involved in lipogenesis and the alteration of energy balance. Unexpectedly, overexpression of ampd genes affected nitrogen consumption in both N-limited and N-excess media, which resulted in an increase in cell growth and lipid accumulation compared with the control strain when nitrogen was available. Furthermore, the increased lipid accumulation in the ampd-overexpressing mutants in N-excess media was accompanied by enhanced activities of lipid biosynthetic enzymes. These data suggested that nitrogen metabolism and energy metabolism are affected by AMPD, and overexpression of ampd genes induced lipid accumulation under nitrogen-rich conditions by mimicking the nitrogen limitation response. This highlights an intriguing function of AMPD in M. circinelloides.

Enhanced reducing sugar production and extraction for Chlorella vulgaris in mixotrophic cultivation using high hydrostatic pressure processing and ultrasound

ABSTRACT Although extraction of polysaccharides to convert reducing sugars (RS) from microalgae by acid or alkali pretreatments and enzymatic hydrolysis has been extensively studied, few reports exploring the use of high hydrostatic pressure processing (HHP) and ultrasonication (US) as emerging technologies for the extraction of sugars from microalgae biomass exist. Thus, the present study was conducted to determine the effects of mixotrophic growth and stress conditions (NaNO3 and CO2 concentration and light intensity) on RS and protein accumulation in the unicellular green alga Chlorella vulgaris in addition to optimization of the effectiveness of the sequential applications of HHP and US with dilute acid as well as simultaneous enzymatic saccharification on the production of RS from microalga cells. High light intensity, high CO2 concentration and limited nitrogen concentration promoted RS production. The maximum protein content (0.0683 mg g‒1) was achieved at 0.3 g l‒1 NaNO3 concentration, 7000 μmol photons m‒2 s‒1 and 6 l min‒1 CO2 concentration. The highest RS content of C. vulgaris after 48 h enzymatic saccharification (583.86 ± 13.23 mg g‒1) was obtained at 1% (w/w) acid concentration and 80% amplitude for 30 min with 79.4% RS yield. Combined US-assisted dilute acid pretreatment and enzymatic hydrolysis were also found to be more effective than HHP assisted dilute acid pretreatment and enzymatic saccharification. Therefore, microalgal biomass can be considered a suitable renewable feedstock used in fermentation.